Modulation of systemic immune responses by transplantation of hematopoietic stem cells transduced with genes encoding antigens and antigen presenting cell regulatory molecules

ABSTRACT

The invention provides methods and compositions for the modulation of systemic immune responses by transplantation of hematopoietic stem cells transduced with genes encoding antigens and antigen presenting cell regulatory molecules. The invention includes bi-cistronic lentiviral expression vectors adapted for antigen expression in antigen presenting cells for use in DNA vaccines directed against pathogens and tumor antigens as well as for the treatment of autoimmune disease and for the establishment of antigen tolerance.

1. BACKGROUND OF THE INVENTION

[0001] Traditional vaccination with immunogenic proteins has eliminatedor reduced the incidence of many diseases in the last century, howeverthere are problems involved in using proteins associated with certainpathogens and disease states. Immunization by genes encoding immunogens,rather than with the immunogen itself, has opened up new possibilitiesfor vaccine research and development and offers chances for newapplications and indications for future vaccines. For example, innumerous animal models, DNA immunization has been shown to induceprotective immunity against infectious diseases (viral, bacterial andprotozoan). Recent examples include DNA vaccine for prophylactic ortherapeutic immunization against hepatitis B virus (Davis (1999) MtSinai J. Med. 66: 84-90). Other applications of DNA vaccine technologyinclude the treatment of cancer as well as auto-immune disease. Forexample, DNA vaccines encoding prostate specific antigen (PSA) have beendeveloped to treat prostate cancer (see e.g. Kim et al. (2001) Oncogene20: 4497-506). Furthermore, DNA vaccine technology has applications intreating autoimmune disease, where the immune system attacks the hostsown tissues, resulting in diseases such as myasthenia gravis, diabetesor multiple sclerosis. Autoimmune disease results from a breakdown intolerance to self antigens; however the same fundamental immunologicalreactions that immunological reactions that control immune responses toforeign antigens also operate in autoimmune diseases (see e.g. Ramshawet al. (1997) Immunol Cell Biol 75: 409-13). Accordingly, DNA vaccinetechnology can be used to reestablish tolerance to self antigens as wellas to establish tolerance to environmental immunogens where desirable.

[0002] These DNA vaccine strategies exploit the underlying mechanisms ofantigen processing, immune presentation and regulation of immuneresponses in order to optimize the prophylactic or therapeutic value ofthe vaccine, particularly for vaccines against chronic or persistentinfectious diseases and tumors. DNA technology has facilitated the rapiddevelopment of plasmid-based vaccines designed to prevent viral,bacterial and parasitic infections. Current DNA vaccination strategiesaddress: the construction of specialized vaccine plasmids; screening forprotective immunogens to be encoded by these plasmids; optimization ofthe mode of application; analysis of vaccine pharmacokinetics; as wellas analysis of vaccine safety and immunotoxicology. DNA vaccines havethe potential to accelerate the research phase of new vaccines and toimprove the chances of success, since finding new immunogens with thedesired properties requires less effort than for conventional vaccines.However, on the way to successful DNA vaccines, several limitations mustbe dealt with including: the persistence and distribution of inoculatedplasmid DNA in vivo; its potential to express antigens inappropriately;and the potentially deleterious ability to insert genes into the hostcell's genome. Patents directed toward and describing such DNA vaccinetechnology include U.S. Pat. Nos. 5,589,466; 5,738,852; 5,925,362;6,130, 052; 6,149,906; 6,214,804; 6,224,870; 6,245,525; and 6,294,378,the contents of which are incorporated herein by reference.

[0003] The initiation of T cell-dependent immune responses depends onpresentation of antigens by bone marrow derived antigen-presenting cells(APCs) such as dendritic cells. Tolerance induction also depends on theexpression of antigens by tolerizing antigen presenting cells.Immunotherapy approaches are aimed at introducing antigen into variouspopulations of APCs such as dendritic cells. However, the efficiencywith which standard immunotherapy and vaccine approaches introducesantigens into APCs is relatively low. Standard vaccines appear tointroduce antigen into relatively small numbers of APCs in the body. Exvivo transduction or loading of dendritic cells followed by theirreinfusion can increase the number of antigen-loaded APCs—however, theex vivo manipulation and maturation of DCs appears to interfere withtheir ability to stimulate T cell responses subsequent to reintroductioninto the body. A second problem solved relates to the importance ofactivating APCs with the appropriate signals. Current approaches utilizereagents provided systemically which have many side effects due to theiractivity on many different cell types. Accordingly, it would bedesirable to have methods and compositions for the effective expressionof antigens in large numbers of bone marrow derived APCs as well as toeffect efficient immune responses to these APCs while avoidingundesirable side effects resulting from strategies employing systemicadministration of immunostimulatory reagents.

SUMMARY OF THE INVENTION

[0004] The invention provides compositions, including vectors such aslentiviral expression vectors, which are used to introduce genesencoding an antigen (the antigen transgene) into hematopoietic stemcells (HSC) ex vivo. The transduced HSCs are then transplanted into asubject to be treated. In preferred embodiments, APC-stimulatory agentsare also delivered to the patient to induce APC (dendritic cell)maturation, expansion and activation. These in vivo differentiated APCsexpress and present the antigen encoded by the transgene introducedoriginally into the hematopoietic stem cells ex vivo. This approachallows for the antigens into APCs systemically.

[0005] In preferred embodiments, the invention provides methods formodulating T cell-dependent immune responses to an antigen in amammalian host. Preferably, such methods transformation or transductionof a population of hematopoietic stem cells by first introducing aconstruct, e.g. a viral expression construct, that expresses two genes.In general, the first gene is a selected antigen, such as a pathogenantigen, a tumor antigen or a self-antigen. The second gene is a factorthat regulates antigen presenting cell differentiation, maturation,expansion or activation. Accordingly, the transduced or transfectedhematopoietic stem cells develop into antigen presenting cells thatexpress the selected antigen either prior to or followingtransplantation into the host organism to be treated. This transgeniccell population thereby expresses the selected antigen and modulatesresponding T cell-dependent immune responses to the selected antigen inthe mammalian host. The population of hematopoietic stem cells may beprovided from an autologous, an allogeneic, or a xenogeneic bone marrowgraft.

[0006] In preferred embodiments, the antigen is a tumor antigen such asa prostate-specific membrane antigen (PSMA), a HER2/neu gene antigen, anidiotypic immunoglobulin antigen, an idiotypic T cell receptor antigen,an SV40 antigen, and a carcinoembryonic antigen (CEA). In theseembodiments, the invention is useful in treating cancers.

[0007] In other embodiment, the antigen is a pathogen antigen such as ahepatitis B antigen, a tuberculosis antigen, an HIV antigen, and aBorrelia burgdorferi sensu lato antigen. In these embodiments theinvention is useful in treating viral and microbial infections.

[0008] In still other embodiments, the antigen is an immunetolerance-inducing antigen such as pancreatic beta-cell antigens,insulin, GAD, collagen type 11, human cartilage gp 39 (HCgp39),gp130-RAPS, myelin basic protein (MBP), proteolipid protein (PLP),myelin oligodendrocyte glycoprotein, fibrillarin, small nucleolarprotein (snoRNP), thyroid stimulating factor receptor (TSH-R), histones,glycoprotein gp70, ribosomal protein, pyruvate dehydrogenasedehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigens andhuman tropomyosin isoform 5 (hTM5). In these embodiments the inventionis useful in treating autoimmune diseases and disorders such as:insulin-dependent diabetes mellitus, rheumatoid arthritis, multiplesclerosis, scleroderma, Graves' disease, systemic lupus erythematosus,primary billiary cirrhosis, alopecia areata; and ulcerative colitis.

[0009] In preferred embodiments the second gene of the transduced ortransfected expression vector encodes a factor that regulates antigenpresenting cell differentiation, maturation, expansion or activationsuch as iCD40-FKBP, iFlt3-FKBP, CD40 ligand, Flt3 ligand, GM-CSF, orIL-12.

[0010] In still other preferred embodiments, the first gene is expressedfrom a dendritic cell-specific promoter such as an HLA-DR promoter, aCIITA P1 promoter, a B7-DC promoter, a “minor” gene promoter, or an EF1a promoter.

[0011] Notably, the method of the invention allows for transplantationof the transgenic cells into the mammalian host either before allowingthem to develop into antigen presenting cells that express the antigen,or after. In preferred embodiments, modulation of the T cell-dependentimmune response effects an immunization against a viral or microbialpathogen.

[0012] In another preferred embodiment, modulation of the Tcell-dependent immune response effects an immune response against atumor antigen. In still other preferred embodiements, modulation of theT cell-dependent immune response effects immune tolerance for anautoantigen.

[0013] In another aspect of the invention, a method for the modulationof a T cell-dependent immune response to an antigen in a mammalian hostis provided. In this aspect of the invention, an expression vectorencoding the first antigen gene is transduced or transfected into apopulation of hematopoietic stem cells and the resulting transgeniccells are contacted with a immunostimulatory factor that regulatesantigen presenting cell differentiation, maturation, expansion oractivation. Following this ex vivo treatment, the treated cells arefurther allowed to differentiate ex vivo or immediately transplantedinto the mammalian host to be treated. The transgenic cells thustransplanted, develop into antigen presenting cells that express theselected antigen and thereby modulate corresponding T cell-dependentimmune responses to the antigen in the mammalian host so as to, e.g.,vaccinate the host against a pathogen, induce an immune response againsta tumor or induce tolerance to an autoantigen.

[0014] In another preferred embodiment, the invention provides vectorexpression system which include a first gene expression cassette thatexpresses an antigen gene under control of an antigen presentingcell-specific promoter, and a second gene expression cassette thatexpresses a factor which stimulates antigen presenting celldifferentiation, maturation, expansion or activation.

[0015] In preferred embodiments, the vector expression system is alentiviral vector and the antigen presenting cell-specific promoter isan HLA-DR promoter; a CIITA P1 promoter; a B7-DC promoter; or a minorgene promoter. In other preferred embodiments, the first gene expressioncassette encodes a pathogen antigen gene such as a hepatitis B antigen,a tuberculosis antigen, an HIV antigen, or a Borrelia burgdorferi sensulato antigen. In other preferred embodiments, the first gene expressioncassette encodes a tumor antigen such as a prostate-specific membraneantigen (PSMA), a HER2/neu gene antigen, an idiotypic immunoglobulinantigen, an idiotypic T cell receptor antigen, an SV40 antigen, or acarcinoembryonic antigen (CEA). In still other preferred embodiments,the first gene expression cassette encodes an immune tolerance-inducingantigen (e.g. an autoantigen gene) such as a pancreatic beta-cellantigen, insulin, GAD, collagen type 11, human cartilage gp 39 (HCgp39),gp130-RAPS, myelin basic protein (MBP), proteolipid protein (PLP),myelin oligodendrocyte glycoprotein, fibrillarin, small nucleolarprotein (snoRNP), thyroid stimulating factor receptor (TSH-R), histones,glycoprotein gp70, ribosomal protein, pyruvate dehydrogenasedehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigens, orhuman tropomyosin isoform 5 (hTM5). In this aspect of the invention, thefactor used to that stimulate antigen presenting cell differentiation,maturation, expansion or activation may be iCD40-PKBP, iFlt3-FKBP, CD40ligand, Flt3 ligand, GM-CSF, or IL-12.

3. BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 depicts lentiviral SIN vectors which allow high levels ofstable gene transfer and regulated transgene expression from a selectivepromoter in transduced cells

[0017]FIG. 2 shows a number of lentiviral vectors expressing theselected antigen and the APC-stimulatory gene.

[0018]FIG. 3 shows that a GFP transgene driven by the DRα promoter isselectively expressed in human HLA-DR+ cells differentiated from CD34⁺cells transduced by the DR.GFPSIN lentiviral vector.

[0019]FIG. 4 shows that transduced human CD34⁺ cells engraft in vivo andgenerate multiple lineages of GFP+ hematopoietic cells including DCsthat can stimulate allogeneic human T cell proliferation.

[0020]FIG. 5 shows that a GFP transgene driven by the DRα promoter isselectively expressed in mouse MHC II+ cells differentiated from bonemarrow cells transduced by the DR.GFP SIN lentiviral vector.

[0021]FIG. 6 shows selective transgene expression mediated by the DRαpromoter in mouse donor-derived DCs post transduction and BMT.

[0022]FIG. 7 shows mouse DCs differentiated from transduced BM cells bythe DR.HA lentiviral vector can specifically and potently stimulate theproliferation of HA-specific T cells (from the 6.5 HA-TCR Tg mice).

[0023]FIG. 8 HA shows transduced HSCs post BMT greatly stimulate theproliferation of HA-specific CD4+ T cells and IFN-y production followingadoptive T cell transfer.

4. DETAILED DESCRIPTION OF THE INVENTION

[0024] 4.1. General

[0025] In general, the invention provides strategies to achieve theexpression of antigens in large numbers of bone marrow derived APCs.This strategy involves the introduction of genes encoding antigen intohematopoietic stem cells ex vivo followed by transplantation of thesetransduced hematopoietic stem cells in vivo. This approach also allowsfor the introduction of genes encoding the signaling portions ofmolecules that regulate APC differentiation and activation. Furthermore,the invention provides approaches for selectively expressing genesencoding antigen and signaling molecules in particular APC subtypesthrough the use of vectors that express the transgenes under control ofinternal promoters that are specifically expressed in particular APCtypes.

[0026] In certain preferred embodiments, the invention provides methodsand compositions for the efficient introduction of antigens into APCs.The invention provides methods and compositions for the ex vivotransduction or loading of dendritic cells followed by their reinfusion,while increasing the APCs ability to stimulate T cell responsessubsequent to reintroduction into the host's body.

[0027] In certain other preferred embodiments, the invention providesmethods and compositions to activate APCs using appropriate signals.While certain approaches to, for example, DNA vaccination utilizereagents provided systemically which have many side effects due to theiractivity on many different cell types, the current invention introducessignaling portions of these molecules into the vector that incorporatesthe antigen under control of promoters specific to particular APC types.Accordingly, in certain embodiments the invention allows for theenhancement of the specificity of the differentiation and activationfunctions of APCs necessary for appropriate T cell stimulation.

[0028] 4.2. Definitions

[0029] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims areprovided below.

[0030] The term “aberrant activity”, as applied to an activity of apolypeptide refers to an activity which differs from the activity of thewild-type or native polypeptide or which differs from the activity ofthe polypeptide in a healthy subject. An activity of a polypeptide canbe aberrant because it is stronger than the activity of its nativecounterpart. Alternatively, an activity can be aberrant because it isweaker or absent relative to the activity of its native counterpart. Anaberrant activity can also be a change in an activity. For example, anaberrant polypeptide can interact with a different target peptide. Acell can have an aberrant polypeptide activity due to overexpression orunderexpression of the gene encoding the polypeptide.

[0031] The term “agonist”, as used herein, is meant to refer to an agentthat mimics or upregulates (e.g. potentiates or supplements) abioactivity. A polypeptide agonist can be a wild-type protein orderivative thereof having at least one bioactivity of the wild-typepolypeptide. A polypeptide therapeutic can also be a compound thatupregulates expression of a polypeptide-encoding gene or which increasesat least one bioactivity of a polypeptide. An agonist can also be acompound which increases the interaction of a polypeptide with anothermolecule, thereby promoting.

[0032] The term “allele”, which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for the gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions, and insertions of nucleotides.Frequently occurring sequence variations include transition mutations(i.e. purine to purine substitutions and pyrimidine to pyrimidinesubstitutions, e.g. A to G or C to T), transversion mutations (i.e.purine to pyrimidine and pyrimidine to purine substitutions, e.g. A to Tor C to G), and alteration in repetitive DNA sequences (e.g. expansionsand contractions of trinucleotide repeat and other tandem repeatsequences). An allele of a gene can also be a form of a gene containinga mutation. The term “allelic variant of a polymorphic region of a FasLgene” refers to a region of a FasL gene having one or several nucleotidesequence differences found in that region of the gene in certainindividuals.

[0033] “Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g. suppresses or inhibits) at least one bioactivity. Anantagonist can be a compound which inhibits or decreases the interactionbetween a protein and another molecule, e.g., a FasL ligand and a FasLreceptor. An antagonist can also be a compound that down-regulatesexpression of a gene or which reduces the amount of gene product proteinpresent. The FasL antagonist can be a dominant negative form of a FasLpolypeptide, e.g., a form of a FasL polypeptide which is capable ofinteracting with a target peptide. An antagonist can also be a compoundthat interferes with a protein-dependent signal transduction pathwaye.g. a dominant negative FADD which blocks downstream FasL signaling.The FasL antagonist can also be a nucleic acid encoding a dominantnegative form of a FasL polypeptide, a FasL antisense nucleic acid, or aribozyme capable of interacting specifically with a FasL RNA. Yet otherFasL antagonists are molecules which bind to a FasL polypeptide andinhibit its action. Such molecules include peptides, e.g., forms of FasLtarget peptides which do not have biological activity, and which inhibitbinding to FasL target molecules, such as the FasL receptor.

[0034] The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments thereof which are also specifically reactive with avertebrate, e.g., mammalian, protein. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. Thus, the termincludes segments of proteolytically-cleaved or recombinantly-preparedportions of an antibody molecule that are capable of selectivelyreacting with a certain protein. Nonlimiting examples of suchproteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv,and single chain antibodies (scFv) containing a V[L] and/or V[H] domainjoined by a peptide linker. The scFv's may be covalently ornon-covalently linked to form antibodies having two or more bindingsites. The subject invention includes polyclonal, monoclonal, or otherpurified preparations of antibodies and recombinant antibodies.

[0035] A disease, disorder, or condition “associated with” or“characterized by” an aberrant expression of a nucleic acid refers to adisease, disorder, or condition in a subject which is caused by,contributed to by, or causative of an aberrant level of expression of anucleic acid.

[0036] As used herein the term “bioactive fragment of a FasLpolypeptide” refers to a fragment of a full-length FasL polypeptide,wherein the fragment specifically mimics or antagonizes the activity ofa wild-type FasL polypeptide. The bioactive fragment preferably is afragment capable of interacting with a FasL receptor.

[0037] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein means an effector or antigenic function that is directly orindirectly performed by a polypeptide (whether in its native ordenatured conformation), or by any subsequence thereof. Biologicalactivities include binding to a target peptide. A target polypeptidebioactivity can be modulated by directly affecting the targetpolypeptide. Alternatively, a target polypeptide bioactivity can bemodulated by modulating the level of the target polypeptide, such as bymodulating expression of the target polypeptide-encoding gene.

[0038] The term “biomarker” refers a biological molecule, e.g., anucleic acid, peptide, hormone, etc., whose presence or concentrationcan be detected and correlated with a known condition, such as a diseasestate.

[0039] “Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0040] A “chimeric polypeptide” or “fusion polypeptide” is a fusion of afirst amino acid sequence encoding one of the subject polypeptides witha second amino acid sequence defining a domain (e.g. polypeptideportion) foreign to and not substantially homologous with any domain ofthe polypeptide. A chimeric polypeptide may present a foreign domainwhich is found (albeit in a different polypeptide) in an organism whichalso expresses the first polypeptide, or it may be an “interspecies”,“intergenic”, etc. fusion of polypeptide structures expressed bydifferent kinds of organisms. In general, a fusion polypeptide can berepresented by the general formula X-polypeptide-Y, wherein“polypeptide” represents a portion or all of a protein of interest and Xand Y are independently absent or represent amino acid sequences whichare not related to the protein sequence in an organism, includingnaturally occurring mutants.

[0041] A “delivery complex” shall mean a targeting means (e.g. amolecule that results in higher affinity binding of a gene, protein,polypeptide or peptide to a target cell surface and/or increasedcellular or nuclear uptake by a target cell). Examples of targetingmeans include: sterols (e.g. cholesterol), lipids (e.g. a cationiclipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associatedvirus, and retrovirus) or target cell specific binding agents (e.g.ligands recognized by target cell specific receptors). Preferredcomplexes are sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex is cleavable under appropriate conditions within the cell sothat the gene, protein, polypeptide or peptide is released in afunctional form.

[0042] The term “dendritic cell” refers to any of various accessorycells that serve as antigen-presenting cells (APCs) in the induction ofan immune response. As used herein, the term “dendritic cell” includesboth interdigitating dendritic cells which are present in theinterstitium of most organs and are abundant in T cell-rich areas of thelymph nodes and spleen, as well as throughout the epidermis of the skin,where they are also referred to as Langerhans cells. The interdigitatingdendritic cells arise from marrow precursor cells and are related inlineage to mononuclear phagocytes.

[0043] As is well known, genes may exist in single or multiple copieswithin the genome of an individual. Such duplicate genes may beidentical or may have certain modifications, including nucleotidesubstitutions, additions or deletions, which all still code forpolypeptides having substantially the same activity. For example, theterm “DNA sequence encoding an antigen polypeptide” may thus refer toone or more antigen genes within a particular individual. Moreover,certain differences in nucleotide sequences may exist between individualorganisms, which are called alleles. Such allelic differences may or maynot result in differences in amino acid sequence of the encodedpolypeptide yet still encode a polypeptide with the same biologicalactivity.

[0044] The term “equivalent” is understood to include nucleotidesequences encoding functionally equivalent polypeptides. Equivalentnucleotide sequences will include sequences that differ by one or morenucleotide substitutions, additions or deletions, such as allelicvariants; and will, therefore, include sequences that differ from thenucleotide sequence of the nucleic acids of the invention due to thedegeneracy of the genetic code.

[0045] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are identical at that position. A degree of homology orsimilarity or identity between nucleic acid sequences is a function ofthe number of identical or matching nucleotides at positions shared bythe nucleic acid sequences. A degree of identity of amino acid sequencesis a function of the number of identical amino acids at positions sharedby the amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of thesequences of the present invention.

[0046] The term “polypeptide binding partner” or “polypeptide BP” refersto various cell proteins which bind to a specified polypeptide of theinvention.

[0047] The term “interact” as used herein is meant to include detectablerelationships or association (e.g. biochemical interactions) betweenmolecules, such as interaction between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid, and protein-small molecule or nucleicacid-small molecule in nature.

[0048] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject polypeptides preferably includes no more than 10 kilobases (kb)of nucleic acid sequence which naturally immediately flanks the subjectgene in genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

[0049] A “knock-in” transgenic animal refers to an animal that has had amodified gene introduced into its genome and the modified gene can be ofexogenous or endogenous origin.

[0050] A “knock-out” transgenic animal refers to an animal in whichthere is partial or complete suppression of the expression of anendogenous gene (e.g, based on deletion of at least a portion of thegene, replacement of at least a portion of the gene with a secondsequence, introduction of stop codons, the mutation of bases encodingcritical amino acids, or the removal of an intron junction, etc.). Inpreferred embodiments, the “knock-out” gene locus corresponding to themodified endogenous gene no longer encodes a functional polypeptideactivity and is said to be a “null” allele. Accordingly, knock-outtransgenic animals of the present invention include those carrying onenull gene mutation, as well as those carrying two null gene mutations.

[0051] A “knock-out construct” refers to a nucleic acid sequence thatcan be used to decrease or suppress expression of a protein encoded byendogenous DNA sequences in a cell. In a simple example, the knock-outconstruct is comprised of a gene with a deletion in a critical portionof the gene so that active protein cannot be expressed therefrom.Alternatively, a number of termination codons can be added to the nativegene to cause early termination of the protein or an intron junction canbe inactivated. In a typical knock-out construct, some portion of thegene is replaced with a selectable marker (such as the neo gene) so thatthe gene can be represented as follows: gene 5′/neo/gene 3′, where gene5′ and gene 3′, refer to genomic or cDNA sequences which are,respectively, upstream and downstream relative to a portion of the geneand where neo refers to a neomycin resistance gene. In another knock-outconstruct, a second selectable marker is added in a flanking position sothat the gene can be represented as: gene/neo/gene/TK, where TK is athymidine kinase gene which can be added to either the gene 5′ or thegene 3′ sequence of the preceding construct and which further can beselected against (i.e. is a negative selectable marker) in appropriatemedia. This two-marker construct allows the selection of homologousrecombination events, which removes the flanking TK marker, fromnon-homologous recombination events which typically retain the TKsequences. The gene deletion and/or replacement can be from the exons,introns, especially intron junctions, and/or the regulatory regions suchas promoters.

[0052] The term “modulation” as used herein refers to both upregulation(i.e., activation or stimulation (e.g., by agonizing or potentiating))and downregulation (i.e. inhibition or suppression (e.g., byantagonizing, decreasing or inhibiting)).

[0053] The term “mutated gene” refers to an allelic form of a gene,which is capable of altering the phenotype of a subject having themutated gene relative to a subject which does not have the mutated gene.If a subject must be homozygous for this mutation to have an alteredphenotype, the mutation is said to be recessive. If one copy of themutated gene is sufficient to alter the genotype of the subject, themutation is said to be dominant. If a subject has one copy of themutated gene and has a phenotype that is intermediate between that of ahomozygous and that of a heterozygous subject (for that gene), themutation is said to be co-dominant.

[0054] The “non-human animals” of the invention include mammalians suchas rodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant genes of the invention is present and/or expressed ordisrupted in some tissues but not others.

[0055] As used herein, the term “nucleic acid” refers to polynucleotidesor oligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and as applicable to the embodiment being described,single (sense or antisense) and double-stranded polynucleotides.

[0056] The term “nucleotide sequence complementary to the nucleotidesequence set forth in SEQ ID No. x” refers to the nucleotide sequence ofthe complementary strand of a nucleic acid strand having SEQ ID No. x.The term “complementary strand” is used herein interchangeably with theterm “complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double stranded nucleic acids, the complement of anucleic acid having SEQ ID No. x refers to the complementary strand ofthe strand having SEQ ID No. x or to any nucleic acid having thenucleotide sequence of the complementary strand of SEQ ID No. x. Whenreferring to a single stranded nucleic acid having the nucleotidesequence SEQ ID No. x, the complement of this nucleic acid is a nucleicacid having a nucleotide sequence which is complementary to that of SEQID No. x. The nucleotide sequences and complementary sequences thereofare always given in the 5′ to 3′ direction.

[0057] The term “percent identical” refers to sequence identity betweentwo amino acid sequences or between two nucleotide sequences. Identitycan each be determined by comparing a position in each sequence whichmay be aligned for purposes of comparison. When an equivalent positionin the compared sequences is occupied by the same base or amino acid,then the molecules are identical at that position; when the equivalentsite occupied by the same or a similar amino acid residue (e.g., similarin steric and/or electronic nature), then the molecules can be referredto as homologous (similar) at that position. Expression as a percentageof homology, similarity, or identity refers to a function of the numberof identical or similar amino acids at positions shared by the comparedsequences. Expression as a percentage of homology, similarity, oridentity refers to a function of the number of identical or similaramino acids at positions shared by the compared sequences. Variousalignment algorithms and/or programs may be used, including FASTA,BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCGsequence analysis package (University of Wisconsin, Madison, Wis.), andcan be used with, e.g., default settings. ENTREZ is available throughthe National Center for Biotechnology Information, National Library ofMedicine, National Institutes of Health, Bethesda, Md. In oneembodiment, the percent identity of two sequences can be determined bythe GCG program with a gap weight of 1, e.g., each amino acid gap isweighted as if it were a single amino acid or nucleotide mismatchbetween the two sequences.

[0058] Other techniques for alignment are described in Methods inEnzymology, vol. 266: Computer Methods for Macromolecular SequenceAnalysis (1996), ed. Doolittle, Academic Press, Inc., a division ofHarcourt Brace & Co., San Diego, Calif., USA. Preferably, an alignmentprogram that permits gaps in the sequence is utilized to align thesequences. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. An alternative search strategy uses MPSRCHsoftware, which runs on a MASPAR computer. MPSRCH uses a Smith-Watermanalgorithm to score sequences on a massively parallel computer. Thisapproach improves ability to pick up distantly related matches, and isespecially tolerant of small gaps and nucleotide sequence errors.Nucleic acid-encoded amino acid sequences can be used to search bothprotein and DNA databases.

[0059] Databases with individual sequences are described in Methods inEnzymology, ed. Doolittle, supra. Databases include Genbank, EMBL, andDNA Database of Japan (DDBJ).

[0060] Preferred nucleic acids have a sequence at least 70%, and morepreferably 80% identical and more preferably 90% and even morepreferably at least 95% identical to an nucleic acid sequence of asequence shown in one of SEQ ID Nos. of the invention. Nucleic acids atleast 90%, more preferably 95%, and most preferably at least about 9899%identical with a nucleic sequence represented in one of the SEQ ID Nos.of the invention are of course also within the scope of the invention.In preferred embodiments, the nucleic acid is mammalian. In comparing anew nucleic acid with known sequences, several alignment tools areavailable. Examples include PileUp, which creates a multiple sequencealignment, and is described in Feng et al., J. Mol. Evol. (1987)25:351-360. Another method, GAP, uses the alignment method of Needlemanet al., J. Mol. Biol. (1970) 48:443-453. GAP is best suited for globalalignment of sequences. A third method, BestFit, functions by insertinggaps to maximize the number of matches using the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math. (1981) 2:482-489.

[0061] The term “polymorphism” refers to the coexistence of more thanone form of a gene or portion (e.g., allelic variant) thereof. A portionof a gene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

[0062] A “polymorphic gene” refers to a gene having at least onepolymorphic region.

[0063] As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

[0064] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

[0065] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding a particular polypeptide is insertedinto a suitable expression vector which is in turn used to transform ahost cell to produce the heterologous protein. Moreover, the phrase“derived from”, with respect to a particular recombinant gene, is meantto include within the meaning of “recombinant protein” those proteinshaving an amino acid sequence of a particular native polypeptide, or anamino acid sequence similar thereto which is generated by mutationsincluding substitutions and deletions (including truncation) of anaturally occurring form of the polypeptide.

[0066] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic (carbon containing) or inorganic molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be screened with any of the assays of the invention to identifycompounds that modulate a bioactivity.

[0067] The term “stem cell” or “hematopoietic stem cell” is meant apluripotent cell of the hematopoietic system capable of differentiatinginto cells of the lymphoid and myeloid lineages.

[0068] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeof the invention to hybridize to at least approximately 6, 12, 20, 30,50, 100, 150, 200, 300, 350, 400 or 425 consecutive nucleotides of avertebrate gene, preferably a mammalian FasL gene.

[0069] The term “transfected stem cell” is meant a stem cell into whichexogenous DNA or an exogenous DNA gene has been introduced by retroviralinfection or other means well known to those of ordinary skill in theart.

[0070] The term “ex vivo gene therapy” is meant the in vitrotransfection or retroviral infection of stem cells to form transfectedstem cells prior to introducing the transfected stem cells into amammal.

[0071] The term “quiescent stem cell” is meant a stem cell in theG.sub.1 or G.sub.0 phase of the cell cycle. A population of cells isconsidered herein to be a population of quiescent cells when at least50%, preferably at least 70%, more preferably at least 80% of the cellsare in the G.sub.1 or G.sub.0 phase of the cell cycle. Quiescent cellsexhibit a single DNA peak by flow-cytometry analysis, a standardtechnique well known to those of ordinary skill in the arts ofimmunology and cell biology. Another technique useful for determiningwhether a population of cells is quiescent is the addition of a chemicalagent to the cell culture medium that is toxic only to actively cyclingcells, i.e., DNA synthesizing cells, and does not kill quiescent cells.Non-exclusive examples of such chemical agents include hydroxyurea andhigh specific activity tritiated thymidine (.sup.3 HtdR). A populationof cells is evaluated as to the percent in an actively cycling state bythe percent of the cell population killed by the chemical agent. A cellpopulation in which in vitro tritiated thymidine killing is less thanapproximately 30%, preferably less than approximately 10%, morepreferably less than approximately 5%, is considered to be quiescent.

[0072] The term “early repopulating stem cells” is meant stem cellswhich are capable of engrafting into the bone marrow of a host mammalwithin approximately 6 weeks post-transplantation.

[0073] The term “late repopulating stem cells,” also termed “long-termrepopulating cells” is meant myelolymphoid stem cells which are capableof engrafting into the bone marrow of a host mammal after approximately6 weeks post-transplantation.

[0074] By the term “engrafting” or engraftment” is meant the persistenceof proliferating stem cells in a particular location over time. Thus,early repopulating stem cells do not persist for more than about 6weeks, whereas late repopulating stem cells persist for longer, andpreferably much longer, than about 6 weeks.

[0075] Cycling stem cells can be treated to become quiescent by serum orisoleucine starvation. Quiescence can also be induced by reduction ofnutrients in the culture medium such that the cycling stem cells enterand remain in the G.sub.1 or G.sub.0 phase of the cell cycle while thenutrient level is reduced. These methods can be used alone or incombination.

[0076] By the term “expanded population” is meant a population of cells,wherein at least 50% of the cells have divided at least once. In certainembodiments of the invention, the cells may be induced to divide by theadministration of cell cycling agents such as 5-FU and/or cytokines suchas IL-3-CHO, IL-6, IL-11, and other growth stimulating factors wellknown to those of ordinary skill in the art of immunology.

[0077] By the term “non-myeloablated host mammal” is meant a mammalwhich has not undergone irradiation, or other treatment (such aschemical treatment) well known to those of ordinary skill in the art, tocause the death of the bone marrow cells of the mammal.

[0078] By the term “myeloablated host mammal” is meant a mammal whichhas undergone irradiation, or other treatment, such as chemicaltreatment, well known to those of ordinary skill in the art, to causethe death of at least 50% of the bone marrow cells of the mammal.

[0079] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. In preferred embodiments, transcription of one of the FasL genesis under the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of FasL polypeptide.

[0080] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a polypeptide of theinvention (e.g. a gene encoding an antigen or an APC immunostimulatoryactivity) or, in the case of antisense expression from the transferredgene, the expression of a naturally-occurring form of the particulartarget polypeptide is disrupted.

[0081] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the antigen or APC immunostimulatorypolypeptides, or an antisense transcript thereto) which has beenintroduced into a cell. A transgene could be partly or entirelyheterologous, i.e., foreign, to the transgenic animal or cell into whichit is introduced, or, is homologous to an endogenous gene of thetransgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A transgene canalso be present in a cell in the form of an episome. A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

[0082] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of a polypeptide for use in the invention, e.g. eitheragonistic or antagonistic forms. However, transgenic animals in which arecombinant target gene is silent are also contemplated, as for example,the FLP or CRE recombinase dependent constructs described below.Moreover, “transgenic animal” also includes those recombinant animals inwhich gene disruption of one or more target genes is caused by humanintervention, including both recombination and antisense techniques.

[0083] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the condition ordisease.

[0084] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

[0085] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

[0086] 4.3. Nucleic Acids of the Present Invention

[0087] The invention provides antigen-encoding and APC-stimulatoryfactor-encoding and other nucleic acids, homologs thereof, and portionsthereof. Preferred nucleic acids have a sequence at least about 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, and more preferably 85% homologous andmore preferably 90% and more preferably 95% and even more preferably atleast 99% homologous with a nucleotide sequence of a subject gene, e.g.,an antigen-encoding gene Nucleic acids at least 90%, more preferably95%, and most preferably at least about 98-99% identical with a nucleicsequence represented in one of the subject nucleic acids of theinvention or complement thereof are of course also within the scope ofthe invention. In preferred embodiments, the nucleic acid is mammalianand in particularly preferred embodiments, includes all or a portion ofthe nucleotide sequence corresponding to the coding region whichcorrespond to the coding sequences of the subject antigen-encoding DNAs.

[0088] The invention also pertains to isolated nucleic acids comprisinga nucleotide sequence encoding antigen polypeptides, variants and/orequivalents of such nucleic acids. The term equivalent is understood toinclude nucleotide sequences encoding functionally equivalent antigenpolypeptides or functionally equivalent peptides having an activity ofan antigen protein such as described herein. Equivalent nucleotidesequences will include sequences that differ by one or more nucleotidesubstitution, addition or deletion, such as allelic variants; and will,therefore, include sequences that differ from the nucleotide sequencesof e.g. the corresponding antigen gene GenBank entries due to thedegeneracy of the genetic code.

[0089] Preferred nucleic acids are vertebrate antigen nucleic acids.Particularly preferred vertebrate antigen nucleic acids are mammalian.Regardless of species, particularly preferred antigennucleic acidsencode polypeptides that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%,80%, 90%, or 95% similar or identical to an amino acid sequence of avertebrate antigenprotein. In one embodiment, the nucleic acid is a cDNAencoding a polypeptide having at least one bio-activity of the subjectantigen polypeptides or APC-stimulatory factors. Preferably, the nucleicacid includes all or a portion of the nucleotide sequence correspondingto the nucleic acids available through GenBank.

[0090] Still other preferred nucleic acids of the present inventionencode an antigen-encoding polypeptide which is comprised of at least 2,5, 10, 25, 50, 100, 150 or 200 amino acid residues. For example, suchnucleic acids can comprise about 50, 60, 70, 80, 90, or 100 base pairs.Also within the scope of the invention are nucleic acid molecules foruse as probes/primer or antisense molecules (i.e. noncoding nucleic acidmolecules), which can comprise at least about 6, 12, 20, 30, 50, 60, 70,80, 90 or 100 base pairs in length.

[0091] Another aspect of the invention provides a nucleic acid whichhybridizes under stringent conditions to a nucleic acid represented byany of the subject nucleic acids of the invention. Appropriatestringency conditions which promote DNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0× SSC at 50° C., are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6 or in Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press (1989). For example, the salt concentration in thewash step can be selected from a low stringency of about 2.0× SSC at 50°C. to a high stringency of about 0.2× SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C. Both temperature and salt may be varied, ortemperature and salt concentration may be held constant while the othervariable is changed. In a preferred embodiment, an antigen nucleic acidof the present invention will bind to one of the subject SEQ ID Nos. orcomplement thereof under moderately stringent conditions, for example atabout 2.0× SSC and about 40° C. In a particularly preferred embodiment,an antigen-encoding nucleic acid of the present invention will bind toone. of the nucleic acid sequences of FIG. 8A or 9A or complementthereof under high stringency conditions. In another particularlypreferred embodiment, an antigen-encoding nucleic acid sequence of thepresent invention will bind to one of the nucleic acids of the inventionwhich correspond to an antigen-encoding ORF nucleic acid sequences,under high stringency conditions.

[0092] Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of the nucleic acids of the invention orcomplement thereof due to degeneracy in the genetic code are also withinthe scope of the invention. Such nucleic acids encode functionallyequivalent peptides (i.e., peptides having a biological activity of anantigen-encoding polypeptide) but differ in sequence from the sequenceshown in the sequence listing due to degeneracy in the genetic code. Forexample, a number of amino acids are designated by more than onetriplet. Codons that specify the same amino acid, or synonyms (forexample, CAU and CAC each encode histidine) may result in “silent”mutations which do not affect the amino acid sequence of anAntigenpolypeptide. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject antigen polypeptides will exist among mammals. One skilled inthe art will appreciate that these variations in one or more nucleotides(e.g., up to about 3-5% of the nucleotides) of the nucleic acidsencoding polypeptides having an activity of an antigen-encodingpolypeptide may exist among individuals of a given species due tonatural allelic variation.

[0093] 4.3.1 Probes and Primers

[0094] The nucleotide sequences determined from the cloning ofantigengenes from mammalian organisms will further allow for thegeneration of probes and primers designed for use in identifying and/orcloning other antigen homologs in other cell types, e.g., from othertissues, as well as antigen homologs from other mammalian organisms. Forinstance, the present invention also provides a probe/primer comprisinga substantially purified oligonucleotide, which oligonucleotidecomprises a region of nucleotide sequence that hybridizes understringent conditions to at least approximately 12, preferably 25, morepreferably 40, 50 or 75 consecutive nucleotides of sense or anti-sensesequence selected from one of the nucleic acids (e.g. anantigen-encoding nucleic acid) of the invention.

[0095] In preferred embodiments, the antigen primers are designed so asto optimize specificity and avoid secondary structures which affect theefficiency of priming. Optimized PCR primers of the present inventionare designed so that “upstream” and “downstream” primers haveapproximately equal melting temperatures such as can be estimated usingthe formulae: T_(m)=81.5° C.-16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(%formamide)−(600/length); or T_(m)(° C.)=2(A/T)+4(G/C). OptimizedAntigenprimers may also be designed by using various programs, such as“Primer3” provided by the Whitehead Institute for Bi

[0096] Likewise, probes based on the subject antigen sequences can beused to detect transcripts or genomic sequences encoding the same orhomologous proteins, for use, e.g, in prognostic or diagnostic assays(further described below). The invention provides probes which arecommon to alternatively spliced variants of the antigentranscript, suchas those corresponding to at least 12 consecutive nucleotidescomplementary to a sequence found in any of the gene sequences of theinvention. In addition, the invention provides probes which hybridizespecifically to alternatively spliced forms of the antigen transcript.Probes and primers can be prepared and modified, e.g., as previouslydescribed herein for other types of nucleic acids.

[0097] 4.3.3. Antigens

[0098] The invention provides for antigens and antigens andantigen-expressing genes for use in the invention as described below.

[0099] 4.3.3.1 Pathogen Antigens for Immunization Against InfectiousDisease

[0100] Where the antigen encoded by the transduced expression vector isa pathogen antigen, such as a bacterial or viral antigen, the inventionallows for the treatment and protection against infectious disease—i.e.in traditional DNA vaccine applications. Numerous pathogen antigens foruse in this aspect of the invention are known in the art and may beobtained using e.g. standard cloning techniques and/or the nucleic acidand polypeptide sequence information provided in GenBank and othersources (see e.g. www.ncbi.nlm.nih.gov/entrez).

[0101] Exemplary pathogen antigens for use in the invention include:hepatitis B antigen (e.g. HBcAg or the secreted form HBeAg of the coreprotein of hepatitis B virus (HBV), see e.g. Kuhrober (1997) Int Immunol9: 1203-12) for use in treating and preventing hepatitis B infection;tuberculosis antigen for use in treating and preventing tuberculosis(see e.g. Montgomery (2000) Brief Bioinform 1: 289-96); HIV antigen(e.g. gp160) for use in treating and preventing HIV infections (see e.g.Schultz et al. (2000) Intervirology 43: 197-217); and Borreliaburgdorferi sensu lato antigens (e.g. outer surface lipoprotein A(OspA)) for treating and preventing Lyme disease (see e.g. Simon et al.(1999) Zentralbl Bakteriol 289: 690-5). Moreover, the sequencing ofbacterial genomes and subsequent identification of surface-exposedmicrobial structures and their conservation in natural populations ofpathogenic species allows for the rapid identification of primecandidates for many additional pathogen antigens for use in theinvention (see e.g. Saunder and Moxon (1998) Curr Opin Biotechnol 9:618-23).

[0102] 4.3.3.2 Tumor Antigens for Treatment of Cancers

[0103] Where the antigen encoded by the transduced expression vector isa tumor antigen, the invention allows for the treatment and protectionagainst cancers. Numerous tumor antigens for use in this aspect of theinvention are known in the art and may be obtained using e.g. standardcloning techniques and/or the nucleic acid and polypeptide sequenceinformation provided in GenBank and other sources (see e.g.www.ncbi.nlm.nih.gov/entrez).

[0104] Exemplary tumor antigens for use in the invention include: theprostate-specific membrane antigen (PSMA) to treat prostate cancer (seee.g. Mincheff et al. (2000) Eur Urol 38: 208-17); the HER2/neu geneantigen to treat breast cancer (see e.g. Lachman et al. (2001) CancerGene Ther 8: 259-68); idiotypic immunoglobulin sequences to treat B-cellmalignancies (see see e.g. Stevenson et al. (2001) Ann Hematol 80 suppl3: B132-4); idiotypic T cell receptor antigens to treat T cellmalignancies (see e.g. Reddy et al. (2001) Ann NY Acad Scie 941:97-105); an SV40 tumor antigen to treat SV40-expressing tumors (see e.g.Watts et al. (2000) Dev Biol (Basel) 104: 143-7); and carcinoembryonicantigen (CEA) and CD40 ligand antigen to treat carcinomas (see e.g.Xiang et al. (2001) J Immunol 167: 4560-5).

[0105] Also included are fusions of such tumor antigens to antigenicpolypeptides (e.g. tetanus toxin polypeptides see e.g. Stevenson et al.(2001) Ann Hematol 80 suppl 3: B1324) to increase the immune response tothe tumor antigen.

[0106] 4.3.3.3 Auto-antigens for Treatment of Autoimmune Disease

[0107] Where the antigen encoded by the transduced expression vector isan autoantigen, the invention allows for the induction of tolerance tothe autoantigen for the treatment of autoimmune disease. For example,autoantibodies directed against the acetylcholine receptor (AChR) areobserved in patients with Myasthenia gravis, and, accordingly,AchR-antigen expressing vectors may be used in the invention to treatand prevent Myasthenia gravis.

[0108] Notably, even in diseases where the pathogenic autoantigen isunknown, bystander suppression may be induced using antigens present inthe anatomical vicinity. For example, autoantibodies to collagen areobserved in rheumatoid arthritis and, accordingly, a collagen-encodinggene may be utilized as the antigen-expressing gene module in order totreat rheumatoid arthritis (see e.g. Choy (2000) Curr Opin InvestigDrugs 1: 58-62). Furthermore, tolerance to beta cell autoantigens may beutilized to prevent development of type 1 diabetes (see e.g. Bach andChatenoud (2001) Ann Rev Immunol 19: 131-161).

[0109] As another example, auto-antibodies directed against myelinoligodendrocyte glycoprotein (MOG) is observed in autoimmuneencephalomyelitis and in many other CNS diseases as well as multiplesclerosis (see e.g. Iglesias et al. (2001) Glia 36: 22-34). Accordingly,use of MOG antigen expressing constructs in the invention allows fortreatment of multiple sclerosis as well as related autoimmune disordersof the central nervous system.

[0110] Still other examples of candidate autoantigens for use intreating autoimmune disease include: pancreatic beta-cell antigens,insulin and GAD to treat insulin-dependent diabetes mellitus; collagentype 11, human catilage gp 39 (HCgp39) and gp130-RAPS for use intreating rheumatoid arthritis; myelin basic protein (MBP), proteolipidprotein (PLP) and myelin oligodendrocyte glycoprotein (MOG, see above)to treat multiple sclerosis; fibrillarin, and small nucleolar protein(snoRNP) to treat scleroderma; thyroid stimulating factor receptor(TSH-R) for use in treating Graves' disease; nuclear antigens, histones,glycoprotein gp70 and ribosomal proteins for use in treating systemiclupus erythematosus; pyruvate dehydrogenase dehydrolipoamideacetyltransferase (PCD-E2) for use in treating primary billiarycirrhosis; hair follicle antigens for use in treating alopecia areata;and human tropomyosin isoform 5 (hTM5) for use in treating ulcerativecolitis.

[0111] 4.4. APC-Stimulatory Factors

[0112] Various cytokines and other molecules can stimulate the growth,differentiation, migration, and activation of dendritic cells or otherantigen presenting cells and can also boost the ability of dendriticcells to trigger and enhance T cell responses to antigen presentation.See, e.g., Banchereau J et al., “Dendritic cells and the control ofimmunity.” Nature (1998) 392: 245-52; Young J W et al., “Thehematopoietic development of dendritic cells: a distinct pathway formyeloid differentiation.” Stem Cells, (1996) 14:376-387; Cella M et al.,“Origin, maturation and antigen presenting function of dendritic cells.”Curr Opin Immunol. (1997) 9:10-16; Curti A et al., “Dendritic celldifferentiation from hematopoietic CD34⁺ progenitor cells. J. Biol.Regul. Homeost. Agents (2001) 15:49-52.

[0113] Examples of molecules that can modulate differentiation,maturation, expansion or activation of dendritic cells or other antigenpresenting cells include ligands such as CD40 ligand,granulocyte-macrophage colony stimulating factor (GM-CSF), FMS-likereceptor tyrosine kinase 3 ligand (Flt3 ligand, FL), interleukin (IL)I-alpha, IL 1-beta, IL-3, IL-4, IL-6, IL-12, IL-13, IL-15, tumornecrosis factor alpha (TNF-α), granulocyte colony stimulating factor(G-CSF), stem cell factor (SCF, also known as kit ligand, KL, SteelFactor, SF, SLF, and Mast cell growth factor, MGF), tumor necrosisfactor (TNF)-related activation-induced cytokine (TRANCE), and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL), andtransforming growth factor β1. Fusion proteins having one or moreactivities ascribed to any of the above molecules may also modulatedifferentiation, maturation, expansion, or activation of dendritic cellsor other antigen presenting cells. Any of these ligands, fusionproteins, or other molecules could be encoded as a second geneexpression cassette in a vector expression system.

[0114] CD40 ligand has been reported to promote induction of dendriticcells and facilitate development of immunogenic responses. See, e.g.,Borges L et al., “Synergistic action of fms-like tyrosine kinase 3ligand and CD40 ligand in the induction of dendritic cells andgeneration of antitumor immunity in vivo.” J Immunol. (1999)163:1289-1297; Grewal I, Flavell R. “The CD40 ligand. At the center ofthe immune universe?” Immunol Res. (1997)16:59-70. Exemplary nucleicacids that encode CD40 ligand and equivalents are described (see, e.g.Genbank accession nos. X65453 and L07414), as are preparations,compositions, and methods of use (U.S. Pat. No. 6,290,972 to Armitage etal.)

[0115] GM-CSF (for exemplary nucleic acids encoding GM-CSF andequivalents, see, e.g., Genbank accession nos. X03020, X03019, X03221,E02975, E02287, E01817, E00951, E00950, A20083, A11763, and X03021) hasbeen reported modulate mobilization, differentiation, expansion, andactivation of dendritic cells and other antigen presenting cells. See,e.g., Arpinati M et al., “Granulocyte-colony stimulating factormobilizes T helper 2-inducing dendritic cells.” Blood. (2000)95(8):2484-2490; Pulendran B et al., “Flt3-ligand and granulocytecolony-stimulating factor mobilize distinct human dendritic cell subsetsin vivo.” J Immunol. (2000) 165(1):566-572; Sallusto F, Lanzavecchia A,“Efficient presentation of soluble antigen by cultured human dendriticcells is maintained by granulocyte/macrophage colony-stimulating factorplus interleukin 4 and down-regulated by tumor necrosis factor α.” J ExpMed (1994) 182: 389-400; Szabolcs P et al., “Expansion ofimmunostimulatory dendritic cells among the myeloid progeny of humanCD34⁺ bone marrow precursors cultured with c-kit ligand,granulocyte-macrophage colony-stimulating factor, and TNF-α.” J Immunol(1995) 154: 5851-61; Caux C et al., “Tumor necrosis factor α stronglypotentiates interleukin-3 and granulocyte-macrophage colony-stimulatingfactor-induced proliferation of human CD34⁺ hematopoietic progenitorcells.” Blood (1990) 75: 2292-8. Compositions, preparations, methods ofmanufacture and use, analogs, fusions, and equivalents ofGM-CSF-encoding exemplary nucleic acid are described, e.g., in U.S. Pat.Nos. 5,641,663, 5,908,763, 5,891,429, 5,393,870, 5,073,627, 5,359,035,and in foreign patent documents JP 1991155798, JP 1990076596, JP1989020097, GB 2212160, EP 0352707, EP 0228018, and WO8504188).

[0116] Flt3 ligand has been described to modulate mobilization,induction, and proliferation of dendritic and other antigen presentingcells. See, e.g., Pulendran B et al., “Flt3-ligand and granulocytecolony-stimulating factor mobilize distinct human dendritic cell subsetsin vivo.” J Immunol. (2000) 165(1):566-572; Borges L et al.,“Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligandin the induction of dendritic cells and generation of antitumor immunityin vivo.” J Immunol. (1999) 163:1289-1297; Lebsack M et al., “Safety ofFLT3 ligand in healthy volunteers.” Blood (1997) 90(Suppl. 1, Abstract751): 170 a; Lyman S D. Biologic effects and potential clinicalapplications of Flt3 ligand. Curr Opin Hematol. (1998) 5(3): 192-196;Maraskovsky E et al., “Dramatic increase in the numbers of functionallymature dendritic cells in FLT3-ligand-treated mice: multiple dendriticcell subpopulations identified.” J Exp Med (1996) 184: 1953-62; StroblH, et al., “Flt3-ligand in cooperation with transforming growthfactor-β1 potentiates in vitro development of Langherans-type dendriticcells and allows single-cell dendritic cell cluster formation underserum-free conditions.” Blood (1997) 90: 1425-34. Exemplary nucleicacids encoding Flt3 ligand and equivalents are disclosed, e.g., inGenbank accession nos. NM_(—)013520, L23636, U04807, U44024, U29875,U03858, U29874, and U04806). Preparations, compositions, and methods ofuse are described, e.g., in U.S. Pat. Nos. 6,291,661, 5,843,423, and5,554,512.

[0117] Exemplary nucleic acids encoding IL-12 and equivalents aredescribed, e.g., in Genbank accession nos. AF401989, AF411293, AF180563,AF180562, AF101062, AY008847, XM_(—)084136, M65271, AF050083,XM_(—)004011, M86672, NM_(—)008351, M86671, and NM_(—)008352 and in U.S.Pat. No. 5,723,127 to Scott et al.

[0118] TNF-α has been found to affect multiple aspects of dendritic cellproliferation and development. See, e.g., Szabolcs P et al., “Expansionof immunostimulatory dendritic cells among the myeloid progeny of humanCD34⁺ bone marrow precursors cultured with c-kit ligand,granulocyte-macrophage colony-stimulating factor, and TNF-α.” J Immunol(1995) 154: 5851-61; Caux C et al., “Tumor necrosis factor α stronglypotentiates interleukin-3 and granulocyte-macrophage colony-stimulatingfactor-induced proliferation of human CD34⁺ hematopoietic progenitorcells.” Blood (1990) 75: 2292-8; Chen B et al., “The role of tumornecrosis factor (in modulating the quantity of peripheral blood-derived,cytokine-driven human dendritic cells and its role in enhancing thequality of dendritic cell function in presenting soluble antigens toCD4+ T cells in vitro.” Blood. (1998) 91(12):4652-4661. Exemplarynucleic acids encoding TNF-α and equivalents are disclosed, e.g., inGenbank accession nos. X01394, A21522, NM_(—)013693, M20155, M38296, andMI 1731, and in U.S. Pat. Nos. 4,677,063, 4,677,064, 4,677,197, and5,298,407.

[0119] TRANCE has been reported to increase survival andimmunostimulatory properties of dendritic cells. See, e.g., Josien F etal., “TRANCE, a tumor necrosis factor family member enhances thelongevity and adjuvant properties of DCs in vivo.” J Exp Med. 2000;191(3):495-502. Exemplary nucleic acids encoding TRANCE and equivalentsare disclosed, e.g., in Genbank accession nos. NM_(—)011613, AF013170,NM_(—)033012, NM_(—)003701, AF053712, AF013171, and AB037599, and inU.S. Pat. No. 6,242,586.

[0120] TRAIL has been shown to promote the ability of dendritic cells tocause apoptosis-of tumor cells targets. See, e.g., Fanger N A,Maliszewski C R, Schooley K, Griffith T S. Human dendritic cells mediatecellular apoptosis via tumor necrosis factor-related apoptosis-inducingligand (TRAIL). J Exp Med. 1999;190(8):1155-1164. Exemplary nucleicacids encoding TRAIL and equivalents are disclosed, e.g., in Genbankaccession nos. U37518, NM_(—)003810 XM_(—)045049, U37522, NM_(—)009425,and AB052771, and in U.S. Pat. No. 5,763,223.

[0121] Exemplary nucleic acids encoding G-CSF and equivalents aredisclosed, e.g., in Genbank accession nos. M17706, X03655, X03438,X03656, M13926, NM_(—)009971, and X05402, and in U.S. Pat. No.4,810,643, and in foreign patent documents WO-A8702060, WO-A-8604605,and WO-A-8604506.

[0122] Exemplary nucleic acids encoding IL-4 and equivalents aredisclosed, e.g., in Genbank accession nos. NM_(—)000589, M13982, X81851,AF395008, M23442, NM_(—)021283, M25892, X05064, X05253, and X05252, andin U.S. Pat. No. 5,017,691. See also Tarte K, Klein B. Dendriticcell-based vaccine: a promising approach for cancer immunotherapy.Leukemia. 1999; 13:653-663.

[0123] c-Kit ligand has been shown to support proliferation andlong-term maintenance of dendritic cells, especially in synergy withother factors. See, e.g., Szabolcs P et al., “Expansion ofimmunostimulatory dendritic cells among the myeloid progeny of humanCD34⁺ bone marrow precursors cultured with c-kit ligand,granulocyte-macrophage colony-stimulating factor, and TNF-α.” J Immunol(1995)154: 5851-61. Exemplary nucleic acids encoding kit ligand andequivalents are disclosed, e.g., in Genbank accession nos. AF400437,AF400436, M59964, M59964, NM_(—)000899, NM_(—)003994, and U44725, and inU.S. Pat. Nos. 6,001,803 and 5,525,708.

[0124] Exemplary nucleic acids encoding IL-13 and equivalents aredisclosed, e.g., in Genbank accession nos. NM_(—)002188, X69079, L06801,U10307, AF377331, NM_(—)008355, L13028, and M23504, and in U.S. Pat.Nos. 5,652,123 and 5,696,234.

[0125] Exemplary nucleic acids encoding IL-1a and equivalents aredisclosed, e.g., in Genbank accession nos. NM_(—)000575, M28983, X02531,M15329, AF010237, NM_(—)013598, M57647, and X68989, and in U.S. Pat.Nos. 5,371,204, 5,008,374, 5,017,692, and 5,756,675.

[0126] Exemplary nucleic acids encoding IL-6 and equivalents aredisclosed, e.g., in Genbank accession nos. X02532, M15330, and M15840,and in U.S. Pat. Nos. 5,286,847 and 5,047,505.

[0127] Exemplary nucleic acids encoding IL-6 and equivalents aredisclosed, e.g., in Genbank accession nos. Y00081, X04602, M54894,M38669, and M14584, and in U.S. Pat. No. 5,338,834.

[0128] Exemplary nucleic acids encoding IL-15 and equivalents aredisclosed, e.g., in Genbank accession nos. U14407, NM_(—)000585, X91233,Z38000, X94222, Y09908, U14332, NM_(—)008357, and AF038164, and in U.S.Pat. No. 5,747,024.

[0129] Exemplary nucleic acids encoding TGF-β1 and equivalents aredisclosed, e.g., in Genbank accession nos. M38449, M55656, X05839,Y00112, X02812, J05114, AJ009862, M13177, and BC013738. See also, e.g.,Strobl H et al., “Flt3-ligand in cooperation with transforming growthfactor-β1 potentiates in vitro development of Langherans-type dendriticcells and allows single-cell dendritic cell cluster formation underserum-free conditions.” Blood (1997) 90: 1425-34; Borkowsky T A et al.,“A role for endogenous transforming growth factor-β1 in Langherans cellbiology: the skin of transforming growth factor-β1 null mice is devoidof epidermal Langherans cells.” J. Exp. Med. (1996) 184:4520-30.

[0130] Nucleic acids that encode molecules that block inhibitory signalsare also contemplated for inclusion as Gene 2 in an expression vector.An example of an inhibitory receptor which may be blocked by anantagonist encoded as gene 2 in an exemplary expression vector isvascular endothelial growth factor receptor. See, e.g., Gabrilovich D etal., “Vascular endothelial growth factor inhibits the development ofdendritic cell and dramatically affects the differentiation of multiplehematopoietic lineages in vivo.” Blood 1998; 92: 4150-66.

[0131] Many of the above-mentioned ligands are known to actsynergistically with one another, as described in the references citedabove. Therefore, the present subject matter also contemplatesexpression vector embodiments comprising a tricistronic construct havinga first gene expression cassette comprising an antigen gene undercontrol of an antigen presenting cell-specific promoter, a second geneexpression cassette comprising a factor gene that stimulates antigenpresenting cell differentiation, maturation, expansion or activation,and a third gene expression cassette comprising a factor gene thatstimulates antigen presenting cell differentiation, maturation,expansion or activation, wherein the second and third gene expressioncassettes are any combination of exemplary nucleic acids or theirequivalents encoding any of the exemplary molecules or their equivalentsthat can modulate differentiation, maturation, expansion or activationof dendritic cells or other antigen presenting cells.

[0132] 4.4.1. FKBP-Fusion Proteins

[0133] In certain embodiments, the APC-stimulatory factor is arecombinant fusion protein (e.g. an iCD40-FKBP fusion protein or aniFlt3-FKBP fusion protein) comprising an FKBP protein moiety forcontrolled dimerization. FKBP fusion protein technology for use in theinvention is provided in U.S. patent nos., the contents of which arehereby incorporated herein by reference.

[0134] 4.5. Vectors

[0135] The invention further provides plasmids and vectors encoding anAntigen protein, which can be used to express an Antigen protein in ahost cell. The host cell may be any prokaryotic or eukaryotic cell.Thus, a nucleotide sequence derived from the cloning of mammalianAntigen proteins, encoding all or a selected portion of the full-lengthprotein, can be used to produce a recombinant form of an Antigenpolypeptide via microbial or eukaryotic cellular processes. Ligating thepolynucleotide sequence into a gene construct, such as an expressionvector, and transforming or transfecting into hosts, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells,are standard procedures well known in the art.

[0136] Typically, expression vectors used for expressing, in vivo or invitro an antigen protein contain a nucleic acid encoding an antigenpolypeptide, operably linked to at least one transcriptional regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the subject proteins in the desired fashion (timeand place). Transcriptional regulatory sequences are described inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990).

[0137] Suitable vectors for the expression of an antigen polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

[0138] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook,Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989)Chapters 16 and 17.

[0139] In a preferred embodiment, the promoter is a constitutivepromoter, e.g., a strong viral promoter, e.g., CMV promoter. Thepromoter can also be cell- or tissue-specific, that permits substantialtranscription of the DNA only in predetermined cells, e.g., inprofessional antigen presenting cells, such as a promoter specific forfibroblasts, or smooth muscle cells, retinal cells or RPE cells. Asmooth muscle specific promoter is. e.g. the promoter of the smoothmuscle cell marker SM22alpha (Akyura et al., (2000) Mol Med 6:983.Retinal pigment epithelial cell specific promoter is, e.g., the promoterof the Rpe65 gene (Boulanger et al. (2000) J Biol Chem 275:31274). Thepromoter can also be an inducible promoter, e.g., a metallothioneinpromoter. Other inducible promoters include those that are controlled bythe inducible binding, or activation, of a transcription factor, e.g.,as described in U.S. Pat. Nos. 5,869,337 and 5,830,462 by Crabtree etal., describing small molecule inducible gene expression (a geneticswitch); International patent applications PCT/US94/01617,PCT/US95/10591, PCT/US96/09948 and the like, as well as in otherheterologous transcription systems such as those involvingtetracyclin-based regulation reported by Bujard et al., generallyreferred to as an allosteric “off-switch” described by Gossen and Bujard(Proc. Natl. Acad. Sci. U.S.A. (1992) 89:5547) and in U.S. Pat. Nos.5,464,758; 5,650,298; and 5,589,362 by Bujard et al. Other inducibletranscription systems involve steroid or other hormone-based regulation.The polynucleotide of the invention together with all necessarytranscriptional and translational control sequences is referred toherein as “construct of the invention” or “transgene of the invention.”

[0140] The polynucleotide of the invention may also be introduced intothe cell in which it is to be expressed together with another DNAsequence (which may be on the same or a different DNA molecule as thepolynucleotide of the invention) coding for another agent. Exemplaryagents are further described below. In one embodiment, the DNA encodes apolymerase for transcribing the DNA, and may comprise recognition sitesfor the polymerase and the injectable preparation may include an initialquantity of the polymerase.

[0141] In certain instances, it may be preferred that the polynucleotideis translated for a limited period of time so that the polypeptidedelivery is transitory. This can be achieved, e.g., by the use of aninducible promoter.

[0142] The polynucleotides used in the present invention may also beproduced in part or in total by chemical synthesis, e.g., by thephosphoramidite method described by Beaucage and Carruthers, Tetra.Letts., 22:1859-1862 (1981) or the triester method according to themethod described by Matteucci et al., J. Am. Chem. Soc., 103:3185(1981), and may be performed on commercial automated oligonucleotidesynthesizers. A double-stranded fragment may be obtained from the singlestranded product of chemical synthesis either by synthesizing thecomplementary strand and annealing the strand together under appropriateconditions or by adding the complementary strand using DNA polymerasewith an appropriate primer sequence.

[0143] The polynucleotide of the invention operably linked to allnecessary transcriptional and translational regulation elements can beinjected as naked DNA into a subject. In a preferred embodiment, thepolynucleotide of the invention and necessary regulatory elements arepresent in a plasmid or vector. Thus, the polynucleotide of theinvention may be DNA, which is itself non-replicating, but is insertedinto a plasmid, which may further comprise a replicator. The DNA may bea sequence engineered so as not to integrate into the host cell genome.

[0144] Preferred vectors for use according to the invention areexpression vectors, i.e., vectors that allow expression of a nucleicacid in a cell vectors. Preferred expression vectors are those whichcontain both prokaryotic sequences, to facilitate the propagation of thevector in bacteria, and one or more eukaryotic transcription units thatare expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHygderived vectors are examples of mammalian expression vectors suitablefor transfection of eukaryotic cells. Some of these vectors are modifiedwith sequences from bacterial plasmids, such as pBR322, to facilitatereplication and drug resistance selection in both prokaryotic andeukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook,Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989)Chapters 16 and 17.

[0145] Any means for the introduction of polynucleotides into mammals,human or non-human, may be adapted to the practice of this invention forthe delivery of the various constructs of the invention into theintended recipient. In one embodiment of the invention, the DNAconstructs are delivered to cells by transfection, i.e., by delivery of“naked” DNA or in a complex with a colloidal dispersion system. Acolloidal system includes macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. The preferredcolloidal system of this invention is a lipid-complexed orliposome-formulated DNA. In the former approach, prior to formulation ofDNA, e.g., with lipid, a plasmid containing a transgene bearing thedesired DNA constructs may first be experimentally optimized forexpression (e.g., inclusion of an intron in the 5′ untranslated regionand elimination of unnecessary sequences (Felgner, et al., Ann NY AcadSci 126-139, 1995). Formulation of DNA, e.g. with various lipid orliposome materials, may then be effected using known methods andmaterials and delivered to the recipient mammal. See, e.g., Canonico etal, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol268; Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No.5,679,647 by Carson et al. Colloidal dispersion systems.

[0146] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs, which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0147] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. Naked DNA or DNA associated with adelivery vehicle, e.g., liposomes, can be administered to several sitesin a subject (see below). For example, smooth muscle cells can betargeted with an antibody binding specifically to SM22a, a smooth musclecell marker. Retinal cells and RPE cells can similarly be targeted.

[0148] In a preferred method of the invention, the DNA constructs aredelivered using viral vectors. The transgene may be incorporated intoany of a variety of viral vectors useful in gene therapy, such asrecombinant retroviruses, adenovirus, adeno-associated virus (AAV), andherpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids.While various viral vectors may be used in the practice of thisinvention, AAV- and adenovirus-based approaches are of particularinterest. Such vectors are generally understood to be the recombinantgene delivery system of choice for the transfer of exogenous genes invivo, particularly into humans. The following additional guidance on thechoice and use of viral vectors may be helpful to the practitioner. Asdescribed in greater detail below, such embodiments of the subjectexpression constructs are specifically contemplated for use in variousin vivo and ex vivo gene therapy protocols.

[0149] A. Adenoviral Vectors

[0150] A viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. Knowledge of the geneticorganization of adenovirus, a 36 kB, linear and double-stranded DNAvirus, allows substitution of a large piece of adenoviral DNA withforeign sequences up to 8 kB. In contrast to retrovirus, the infectionof adenoviral DNA into host cells does not result in chromosomalintegration because adenoviral DNA can replicate in an episomal mannerwithout potential genotoxicity. Also, adenoviruses are structurallystable, and no genome rearrangement has been detected after extensiveamplification. Adenovirus can infect virtually all epithelial cellsregardless of their cell cycle stage. So far, adenoviral infectionappears to be linked only to mild disease such as acute respiratorydisease in the human.

[0151] Adenoviruses have been shown in particular to be efficient ingene delivery to the RPE cells. For example, Baffi et al. describe thedelivery of an adenovirus encoding vascular endothelial growth factor tothe subretinal space in the rat, resulting in the expression of VEGF inthe RPE cells of the rat (Baffi et al. (2000) Invest Ophthalmol Vis Sci41:3582). Another reference describes that laser photocoagulationfurther increases the susceptibility of proliferating RPE cells toadenovirus-mediated gene delivery (Lai et al. (1999) Curr Eye Res19:411). Sakamoto et al. describe that a vitrectomy also improvesadenovirus-mediated gene delivery to the retina (Sakamoto et al. (1998)Gene Ther. 5: 1088). Ali et al. report that co-injection of adenovirusexpressing CTLA4-Ig prolongs adenovirally mediated gene expression inthe mouse retina, by blocking T cell activation (Ali et al. (1998) GeneTher. 5:1561). Other references decribing expression of a transgene inretinal cells and RPE cells, upon injection of an adenoviral vectorcomprising the transgene in the vitreous cavity of eyes of non-humananimals include Lai et al. (2000): Invest Ophthalmol Vis Sci 41:580; Yuet al. (2000) Growth Factors 17:301; and Rackoczy et al. (1998) Aust N ZJ Ophthalmol 26 Suppl 1:S56.

[0152] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target-cell range, and high infectivity. Both ends of theviral genome contain 100-200 base pair (bp) inverted terminal repeats(ITR), which are cis elements necessary for viral DNA replication andpackaging. The early (E) and late (L) regions of the genome containdifferent transcription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression, and host cellshut off (Renan (1990) Radiotherap. Oncol. 19:197). The products of thelate genes, including the majority of the viral capsid proteins, areexpressed only after significant processing of a single primarytranscript issued by the major late promoter (MLP). The MLP (located at16.8 m.u.) is particularly efficient during the late phase of infection,and all the mRNAs issued from this promoter possess a 5′ tripartiteleader (TL) sequence which makes them preferred mRNAs for translation.

[0153] The genome of an adenovirus can be manipulated such that itencodes a gene product of interest, but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle (see, forexample, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al.,(1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell68:143-155). Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses can be advantageous in certain circumstances in that theyare not capable of infecting nondividing cells and can be used to infecta wide variety of cell types, including airway epithelium (Rosenfeld etal., (1992) cited supra), endothelial cells (Lemarchand et al., (1992)PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA89:2581-2584).

[0154] Adenovirus vectors have also been used in vaccine development(Grunhaus and Horwitz (1992) Siminar in Virology 3:237; Graham andPrevec (1992) Biotechnology 20:363). Experiments in administeringrecombinant adenovirus to different tissues include trachea instillation(Rosenfeld et al. (1991); Rosenfeld et al. (1992) Cell 68:143), muscleinjection (Ragot et al. (993) Nature 361:647), peripheral intravenousinjection (Herz and Gerard (1993) Proc. Natl. Acad. Sci. U.S.A.90:2812), and stereotactic inoculation into the brain (Le Gal La Salleet al. (1993) Science 254:988).

[0155] Furthermore, the virus particle is relatively stable and amenableto purification and concentration, and as above, can be modified so asto affect the spectrum of infectivity. Additionally, adenovirus is easyto grow and manipulate and exhibits broad host range in vitro and invivo. This group of viruses can be obtained in high titers, e.g.,10⁹-10¹¹ plaque-forming unit (PFU)/ml, and they are highly infective.The life cycle of adenovirus does not require integration into the hostcell genome. The foreign genes delivered by adenovirus vectors areepisomal, and therefore, have low genotoxicity to host cells. No sideeffects have been reported in studies of vaccination with wild-typeadenovirus (Couch et al., 1963; Top et al., 1971), demonstrating theirsafety and therapeutic potential as in vivo gene transfer vectors.Moreover, the carrying capacity of the adenoviral genome for foreign DNAis large (up to 8 kilobases) relative to other gene delivery vectors(Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).Most replication-defective adenoviral vectors currently in use andtherefore favored by the present invention are deleted for all or partsof the viral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkneret al., supra; and Graham et al., in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).Expression of the inserted polynucleotide of the invention can be undercontrol of, for example, the E1A promoter, the major late promoter (MLP)and associated leader sequences, the viral E3 promoter, or exogenouslyadded promoter sequences.

[0156] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in themethod of the present invention. This is because Adenovirus type 5 is ahuman adenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector. As stated above, thetypical vector according to the present invention is replicationdefective and will not have an adenovirus E1 region. Thus, it will bemost convenient to introduce the nucleic acid of interest at theposition from which the E1 coding sequences have been removed. However,the position of insertion of the polynucleotide or construct on theinvention (also referred to as “nucleic acid of interest”) in a regionwithin the adenovirus sequences is not critical to the presentinvention. For example, it may also be inserted in lieu of the deletedE3 region in E3 replacement vectors as described previously by Karlssonet. al. (1986) or in the E4 region where a helper cell line or helpervirus complements the E4 defect.

[0157] A preferred helper cell line is 293 (ATCC Accession No. CRL1573).This helper cell line, also termed a “packaging cell line” was developedby Frank Graham (Graham et al. (1987) J. Gen. Virol. 36:59-72 and Graham(1977) J. General Virology 68:937-940) and provides E1A and E1B intrans. However, helper cell lines may also be derived from human cellssuch as human embryonic kidney cells, muscle cells, hematopoietic cellsor other human embryonic mesenchymal or epithelial cells. Alternatively,the helper cells may be derived from the cells of other mammalianspecies that are permissive for human adenovirus. Such cells include,e.g., Vero cells or other monkey embryonic mesenchymal or epithelialcells.

[0158] Adenoviruses can also be cell type specific, i.e., infect onlyrestricted types of cells and/or express a transgene only in restrictedtypes of cells. For example, the viruses comprise a gene under thetranscriptional control of a transcription initiation regionspecifically regulated by target host cells, as described e.g., in U.S.Pat. No. 5,698,443, by Henderson and Schuur, issued Dec. 16, 1997. Thus,replication competent adenoviruses can be restricted to certain cellsby, e.g., inserting a cell specific response element to regulate asynthesis of a protein necessary for replication, e.g., E1A or E1B.

[0159] DNA sequences of a number of adenovirus types are available fromGenbank. For example, human adenovirus type 5 has GenBank AccessionNo.M73260. The adenovirus DNA sequences may be obtained from any of the42 human adenovirus types currently identified. Various adenovirusstrains are available from the American Type Culture Collection,Rockville, Md., or by request from a number of commercial and academicsources. A transgene as described herein may be incorporated into anyadenoviral vector and delivery protocol, by restriction digest, linkerligation or filling in of ends, and ligation.

[0160] Adenovirus producer cell lines can include one or more of theadenoviral genes E1, E2a, and E4 DNA sequence, for packaging adenovirusvectors in which one or more of these genes have been mutated or deletedare described, e.g., in PCT/US95/15947 (WO 96/18418) by Kadan et al.;PCT/US95/07341 (WO 95/346671) by Kovesdi et al.; PCT/FR94/00624(WO94/28152) by Imler et al.;PCT/FR94/00851 (WO 95/02697) by Perrocaudetet al., PCT/US95/14793 (WO96/14061) by Wang et al.

[0161] B. AAV Vectors

[0162] Yet another viral vector system useful for delivery of thesubject polynucleotides is the adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review, see Muzyczka et al., Curr. Topics in Micro. and Immunol.(1992) 158:97-129).

[0163] AAV has not been associated with the cause of any disease. AAV isnot a transforming or oncogenic virus. AAV integration into chromosomesof human cell lines does not cause any significant alteration in thegrowth properties or morphological characteristics of the cells. Theseproperties of AAV also recommend it as a potentially useful human genetherapy vector.

[0164] AAV is also one of the few viruses that may integrate its DNAinto non-dividing cells, e.g., pulmonary epithelial cells, and exhibitsa high frequency of stable integration (see for example Flotte et al.,(1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al.,(1989) J. Virol. 63:3822-3828; and McLaughlin et al., (1989) J. Virol.62:1963-1973). Vectors containing as little as 300 base pairs of AAV canbe packaged and can integrate. Space for exogenous DNA is limited toabout 4.5 kb. An AAV vector such as that described in Tratschin et al.,(1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA intocells. A variety of nucleic acids have been introduced into differentcell types using AAV vectors (see for example Hermonat et al., (1984)PNAS USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol.4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39;Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993)J. Biol. Chem. 268:3781-3790).

[0165] AAV has been used successfully to introduce gene constructs intoretinal cells in animals, including non-human primates. For example, anAAV virus containing a gene encoding FGF-2 was administered bysubretinal injection into a transgenic rat model for retinitispigmentosa, which resulted in reduction of the rate of photoreceptordegeneration (Lau et al. (2000) Invest. Ophthalmol. Vis. Csci. 41:3622).AAV has been used for gene transduction in photoreceptor cells innon-human animals (see, e.g., Flannery et al. (1997) PNAS 94:6916;Bennett et al. (2000) PNAS 96:9920). RPE cells have also been transducedefficiently by subretinal injection of an AAV (Bennett et al. (1997)Invest. Ophthalmol. Visual Sci. 38:2857). Grant et al. also describethat a recombinant AAV injected into the vitreous body or the subretinalspace of mouse eyes results in the transduction of cells of the retinalpigment epithelium (RPE), ganglion cells and photoreceptor cells for upto three months, i.e., for as long as the experiment was conducted(Grant et al. (1997) Curr. Eye Res. 16, 949). Efficient transduction ofRPE cells in non-human animals is also described in Rollins et al.(2000) Clin Experiment Ophthalmol 28:382-6; Ali et al. (1998) Hum GeneTher 9:81; and Ali et al. (1996) Hum Mol Genet. 5:591.

[0166] The AAV-based expression vector to be used typically includes the145 nucleotide AAV inverted terminal repeats (ITRs) flanking arestriction site that can be used for subcloning of the transgene,either directly using the restriction site available, or by excision ofthe transgene with restriction enzymes followed by blunting of the ends,ligation of appropriate DNA linkers, restriction digestion, and ligationinto the site between the ITRs. The capacity of AAV vectors is about 4.4kb. The following proteins have been expressed using various AAV-basedvectors, and a variety of promoter/enhancers: neomycinphosphotransferase, chloramphenicol acetyl transferase, Fanconi's anemiagene, cystic fibrosis transmembrane conductance regulator, andgranulocyte macrophage colony-stimulating factor (Kotin, R. M., HumanGene Therapy 5:793-801, 1994, Table I). A transgene incorporating thevarious DNA constructs of this invention can similarly be included in anAAV-based vector. As an alternative to inclusion of a constitutivepromoter such as CMV to drive expression of the polynucleotide ofinterest, an AAV promoter can be used (ITR itself or AAV p5 (Flotte, etal. J. Biol. Chem. 268:3781-3790, 1993)).

[0167] Such a vector can be packaged into AAV virions by reportedmethods. For example, a human cell line such as 293 can beco-transfected with the AAV-based expression vector and another plasmidcontaining open reading frames encoding AAV rep and cap (which areobligatory for replication and packaging of the recombinant viralconstruct) under the control of endogenous AAV promoters or aheterologous promoter. In the absence of helper virus, the rep proteinsRep68 and Rep78 prevent accumulation of the replicative form, but uponsuperinfection with adenovirus or herpes virus, these proteins permitreplication from the ITRs (present only in the construct containing thetransgene) and expression of the viral capsid proteins. This systemresults in packaging of the transgene DNA into AAV virions (Carter, B.J., Current Opinion in Biotechnology 3:533-539, 1992; Kotin, R. M, HumanGene Therapy 5:793-801, 1994)). Typically, three days aftertransfection, recombinant AAV is harvested from the cells along withadenovirus and the contaminating adenovirus is then inactivated by heattreatment.

[0168] Methods to improve the titer of AAV can also be used to expressthe polynucleotide of the invention in an AAV virion. Such strategiesinclude, but are not limited to: stable expression of the ITR-flankedtransgene in a cell line followed by transfection with a second plasmidto direct viral packaging; use of a cell line that expresses AAVproteins inducibly, such as temperature-sensitive inducible expressionor pharmacologically inducible expression. Alternatively, a cell can betransformed with a first AAV vector including a 5′ ITR, a 3′ ITRflanking a heterologous gene, and a second AAV vector which includes aninducible origin of replication, e.g., SV40 origin of replication, whichis capable of being induced by an agent, such as the SV40 T antigen andwhich includes DNA sequences encoding the AAV rep and cap proteins. Uponinduction by an agent, the second AAV vector may replicate to a highcopy number, and thereby increased numbers of infectious AAV particlesmay be generated (see, e.g, U.S. Pat. No. 5,693,531 by Chiorini et al.,issued Dec. 2, 1997. In yet another method for producing large amountsof recombinant AAV, a chimeric plasmid is used which incorporate theEpstein Barr Nuclear Antigen (EBNA) gene, the latent origin ofreplication of Epstein Barr virus (oriP) and an AAV genome. Theseplasmids are maintained as a multicopy extra-chromosomal elements incells, such as in 293 cells. Upon addition of wild-type helperfunctions, these cells will produce high amounts of recombinant AAV(U.S. Pat. No. 5,691,176 by Lebkowski et al., issued Nov. 25, 1997). Inanother system, an AAV packaging plasmid is provided that allowsexpression of the rep gene, wherein the p5 promoter, which normallycontrols rep expression, is replaced with a heterologous promoter (U.S.Pat. No. 5,658,776, by Flotte et al., issued Aug. 19, 1997).Additionally, one may increase the efficiency of AAV transduction bytreating the cells with an agent that facilitates the conversion of thesingle stranded form to the double stranded form, as described in Wilsonet al., WO96/39530.

[0169] AAV stocks can be produced as described in Hermonat and Muzyczka(1984) PNAS 81:6466, modified by using the pAAV/Ad described by Samulskiet al. (1989) J. Virol. 63:3822. Concentration and purification of thevirus can be achieved by reported methods such as banding in cesiumchloride gradients, as was used for the initial report of AAV vectorexpression in vivo (Flotte, et al. J. Biol. Chem. 268:3781-3790, 1993)or chromatographic purification, as described in O'Riordan et al.,WO97/08298.

[0170] Methods for in vitro packaging AAV vectors are also available andhave the advantage that there is no size limitation of the DNA packagedinto the particles (see, U.S. Pat. No. 5,688,676, by Zhou et al., issuedNov. 18, 1997). This procedure involves the preparation of cell freepackaging extracts.

[0171] For additional detailed guidance on AAV technology which may beuseful in the practice of the subject invention, including methods andmaterials for the incorporation of a transgene, the propagation andpurification of the recombinant AAV vector containing the transgene, andits use in transfecting cells and mammals, see e.g. Carter et al, U.S.Pat. No. 4,797,368 (10 Jan. 1989); Muzyczka et al, U.S. Pat. No.5,139,941 (18 Aug. 1992); Lebkowski et al, U.S. Pat. No. 5,173,414 (22Dec. 1992); Srivastava, U.S. Pat. No. 5,252,479 (12 Oct. 1993);Lebkowski et al, U.S. Pat. No. 5,354,678 (11 Oct. 1994); Shenk et al,U.S. Pat. No. 5,436,146(25 Jul. 1995); Chatterjee et al, U.S. Pat. No.5,454,935 (12 Dec. 1995), Carter et al WO 93/24641 (published 9 Dec.1993), and Natsoulis, U.S. Pat. No. 5,622,856 (Apr. 22, 1997). Furtherinformation regarding AAVs and the adenovirus or herpes helper functionsrequired can be found in the following articles: Berns and Bohensky(1987), “Adeno-Associated Viruses: An Update”, Advanced in VirusResearch, Academic Press, 33:243-306. The genome of AAV is described inLaughlin et al. (1983) “Cloning of infectious adeno-associated virusgenomes in bacterial plasmids”, Gene, 23: 65-73. Expression of AAV isdescribed in Beaton et al. (1989) “Expression from the Adeno-associatedvirus p5 and p19 promoters is negatively regulated in trans by the repprotein”, J. Virol., 63:4450-4454. Construction of rAAV is described ina number of publications: Tratschin et al. (1984) “Adeno-associatedvirus vector for high frequency integration, expression and rescue ofgenes in mammalian cells”, Mol. Cell. Biol., 4:2072-2081; Hermonat andMuzyczka (1984) “Use of adeno-associated virus as a mammalian DNAcloning vector: Transduction of neomycin resistance into mammaliantissue culture cells”, Proc. Natl. Acad. Sci. USA, 81:6466-6470;McLaughlin et al. (1988) “Adeno-associated virus general transductionvectors: Analysis of Proviral Structures”, J. Virol., 62:1963-1973; andSamulski et al. (1989) “Helper-free stocks of recombinantadeno-associated viruses: normal integration does not require viral geneexpression”, J. Virol., 63:3822-3828. Cell lines that can be transformedby rAAV are those described in Lebkowski et al. (1988) “Adeno-associatedvirus: a vector system for efficient introduction and integration of DNAinto a variety of mammalian cell types”, Mol. Cell. Biol., 8:3988-3996.“Producer” or “packaging” cell lines used in manufacturing recombinantretroviruses are described in Dougherty et al. (1989) J. Virol.,63:3209-3212; and Markowitz et al. (1988) J. Virol., 62:1120-1124.

[0172] C. Hybrid Adenovirus-AAV Vectors

[0173] Hybrid Adenovirus-AAV vectors represented by an adenovirus capsidcontaining a nucleic acid comprising a portion of an adenovirus, and 5′and 3′ ITR sequences from an AAV which flank a selected transgene underthe control of a promoter. See e.g. Wilson et al, International PatentApplication Publication No. WO 96/13598. This hybrid vector ischaracterized by high titer transgene delivery to a host cell and theability to stably integrate the transgene into the host cell chromosomein the presence of the rep gene. This virus is capable of infectingvirtually all cell types (conferred by its adenovirus sequences) andstable long term transgene integration into the host cell genome(conferred by its AAV sequences).

[0174] The adenovirus nucleic acid sequences employed in this vector canrange from a minimum sequence amount, which requires the use of a helpervirus to produce the hybrid virus particle, to only selected deletionsof adenovirus genes, which deleted gene products can be supplied in thehybrid viral process by a packaging cell. For example, a hybrid viruscan comprise the 5′ and 3′ inverted terminal repeat (ITR) sequences ofan adenovirus (which function as origins of replication). The leftterminal sequence (5′) sequence of the Ad5 genome that can be used spansbp 1 to about 360 of the conventional adenovirus genome (also referredto as map units 0-1) and includes the 5′ ITR and the packaging/enhancerdomain. The 3′ adenovirus sequences of the hybrid virus include theright terminal 3′ ITR sequence which is about 580 nucleotides (about bp35,353- end of the adenovirus, referred to as about map units 98.4-100).

[0175] The AAV sequences useful in the hybrid vector are viral sequencesfrom which the rep and cap polypeptide encoding sequences are deletedand are usually the cis acting 5′ and 3′ ITR sequences. Thus, the AAVITR sequences are flanked by the selected adenovirus sequences and theAAV ITR sequences themselves flank a selected transgene. The preparationof the hybrid vector is further described in detail in published PCTapplication entitled “Hybrid Adenovirus-AAV Virus and Method of UseThereof”, WO 96/13598 by Wilson et al.

[0176] For additional detailed guidance on adenovirus and hybridadenovirus-AAV technology which may be useful in the practice of thesubject invention, including methods and materials for the incorporationof a transgene, the propagation and purification of recombinant viruscontaining the transgene, and its use in transfecting cells and mammals,see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, andreferences cited therein.

[0177] D. Retroviruses

[0178] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin (1990)Retroviridae and their Replication” In Fields, Knipe ed. Virology. NewYork: Raven Press). The resulting DNA then stably integrates intocellular chromosomes as a provirus and directs synthesis of viralproteins. The integration results in the retention of the viral genesequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsialproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene, termed psi, functions as asignal for packaging of the genome into virions. Two long terminalrepeat (LTR) sequences are present at the 5′ and 3′ ends of the viralgenome. These contain strong promoter and enhancer sequences and arealso required for integration in the host cell genome (Coffin (1990),supra).

[0179] In order to construct a retroviral vector, a nucleic acid ofinterest is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and psi components is constructed (Mann et al.(1983) Cell 33:153). When a recombinant plasmid containing a human cDNA,together with the retroviral LTR and psi sequences is introduced intothis cell line (by calcium phosphate precipitation for example), the psisequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein (1988) “Retroviral Vectors”, In: Rodriguezand Denhardt ed. Vectors: A Survey of Molecular Cloning Vectors andtheir Uses. Stoneham:Butterworth; Temin, (1986) “Retrovirus Vectors forGene Transfer: Efficient Integration into and Expression of ExogenousDNA in Vertebrate Cell Genome”, In: Kucherlapati ed. Gene Transfer. NewYork: Plenum Press; Mann et al., 1983, supra). The media containing therecombinant retroviruses is then collected, optionally concentrated, andused for gene transfer. Retroviral vectors are able to infect a broadvariety of cell types. Integration and stable expression require thedivision of host cells (Paskind et al. (1975) Virology 67:242). Thisaspect is particularly relevant for the treatment of PVR, since thesevectors allow selective targeting of cells which proliferate, i.e.,selective targeting of the cells in the epiretinal membrane, since theseare the only ones proliferating in eyes of PVR subjects.

[0180] A major prerequisite for the use of retroviruses is to ensure thesafety of their use, particularly with regard to the possibility of thespread of wild-type virus in the cell population. The development ofspecialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding aprotein of the present invention, e.g., a transcriptional activator,rendering the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.A preferred retroviral vector is a pSR MSVtkNeo (Muller et al. (1991)Mol. Cell Biol. 11:1785 and pSR MSV(XbaI) (Sawyers et al. (1995) J. Exp.Med. 181:307) and derivatives thereof. For example, the unique BamHIsites in both of these vectors can be removed by digesting the vectorswith BamHI, filling in with Klenow and religating to produce pSMTN2 andpSMTX2, respectively, as described in PCT/US96/09948 by Clackson et al.Examples of suitable packaging virus lines for preparing both ecotropicand amphotropic retroviral systems include Crip, Cre, 2 and Am.

[0181] Retroviruses, including lentiviruses, have been used to introducea variety of genes into many different cell types, including neuralcells, epithelial cells, retinal cells, endothelial cells, lymphocytes,myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (seefor example, review by Federico (1999) Curr. Opin. Biotechnol. 10:448;Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988)PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018;Armentano et al., (1990) PNAS USA 87:6141-6145; Huber et al., (1991)PNAS USA 88:8039-8043; Ferry et al., (1991) PNAS USA 88:8377-8381;Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al.,(1992) PNAS USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy3:641-647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwu et al.,(1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

[0182] Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan et al., (1992)J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology163:251-254); or coupling cell surface ligands to the viral env proteins(Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be inthe form of the chemical cross-linking with a protein or other variety(e.g. lactose to convert the env protein to an asialoglycoprotein), aswell as by generating fusion proteins (e.g. single-chain antibody/envfusion proteins). This technique, while useful to limit or otherwisedirect the infection to certain tissue types, and can also be used toconvert an ecotropic vector in to an amphotropic vector.

[0183] E. Other Viral Systems

[0184] Other viral vector systems that can be used to deliver apolynucleotide of the invention have been derived from herpes virus,e.g., Herpes Simplex Virus (U.S. Pat. No. 5,631,236 by Woo et al.,issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus(Ridgeway (1988) Ridgeway, “Mammalian expression vectors,” In: RodriguezR L, Denhardt D T, ed. Vectors: A survey of molecular cloning vectorsand their uses. Stoneham: Butterworth,; Baichwal and Sugden (1986)“Vectors for gene transfer derived from animal DNA viruses: Transientand stable expression of transferred genes,” In: Kucherlapati R, ed.Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene,68:1-10), and several RNA viruses. Preferred viruses include analphavirus, a poxivirus, an arena virus, a vaccinia virus, a poliovirus, and the like. They offer several attractive features for variousmammalian cells (Friedmann (1989) Science, 244:1275-1281; Ridgeway,1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988;Horwich et al.(1990) J. Virol., 64:642-650).

[0185] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990, supra).This suggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.(1991) Hepatology, 14:124A).

[0186] Since in certain embodiments, the compositions of the inventionwill be administered via a specific device, e.g., by injection using asyringe, the invention also provides devices, e.g., syringes, comprisinga composition of the invention.

[0187] The preferred mammalian expression vectors include retrovirus andlentivirus based expression vectors, such as those depicted in FIGS. 4Aand 4B. Preferred mammalian vectors typically contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

[0188] In some instances, it may be desirable to express the recombinantANTIGEN polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III)

[0189] When it is desirable to express only a portion of an Antigenprotein, such as a form lacking a portion of the N-terminus, i.e. atruncation mutant which lacks the signal peptide, it may be necessary toadd a start codon (ATG) to the oligonucleotide fragment containing thedesired sequence to be expressed. It is well known in the art that amethionine at the N-terminal position can be enzymatically cleaved bythe use of the enzyme methionine aminopeptidase (MAP). MAP has beencloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757)and Salmonella typhimurium and its in vitro activity has beendemonstrated on recombinant proteins (Miller et al. (1987) PNAS84:2718-1722). Therefore, removal of an N-terminal methionine, ifdesired, can be achieved either in vivo by expressing Antigen derivedpolypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP (e.g., procedure ofMiller et al., supra).

[0190] Moreover, the gene constructs of the present invention can alsobe used as part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of one of the subjectAntigen proteins. Thus, another aspect of the invention featuresexpression vectors for in vivo or in vitro transfection and expressionof an Antigen polypeptide in particular cell types so as to reconstitutethe function of, or alternatively, abrogate the function of Antigen in atissue. This could be desirable, for example, when thenaturally-occurring form of the protein is misexpressed or the naturalprotein is mutated and less active.

[0191] In addition to viral transfer methods, non-viral methods can alsobe employed to cause expression of a subject Antigen polypeptide in thetissue of an animal. Most non-viral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral targeting means of the present invention rely on endocyticpathways for the uptake of the subject Antigen polypeptide gene by thetargeted cell. Exemplary targeting means of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

[0192] In other embodiments transgenic animals, described in more detailbelow could be used to produce recombinant proteins.

[0193] 4.4. Polypeptides of the Present Invention

[0194] The present invention makes available isolated Antigenpolypeptides which are isolated from, or otherwise substantially free ofother cellular proteins. The term “substantially free of other cellularproteins” (also referred to herein as “contaminating proteins”) or“substantially pure or purified preparations” are defined asencompassing preparations of Antigen polypeptides having less than about20% (by dry weight) contaminating protein, and preferably having lessthan about 5% contaminating protein. Functional forms of the subjectpolypeptides can be prepared, for the first time, as purifiedpreparations by using a cloned gene as described herein.

[0195] Preferred Antigen proteins of the invention have an amino acidsequence which is at least about 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, or 95% identicalor homologous to an amino acid sequence of a SEQ ID No. of theinvention, such as a sequence shown in FIG. 8B (SEQ ID No. 2) or 9B (SEQID No. 4). Even more preferred Antigen proteins comprise an amino acidsequence of at least 10, 20, 30, or 50 residues which is at least about70, 80, 90, 95, 97, 98, or 99% homologous or identical to an amino acidsequence of a SEQ ID No. of the invention. Such proteins can berecombinant proteins, and can be, e.g., produced in vitro from nucleicacids comprising a nucleotide sequence set forth in FIG. 8A or 9A, oranother nucleic acid SEQ ID No. of the invention or homologs thereof.For example, recombinant polypeptides preferred by the present inventioncan be encoded by a nucleic acid, which is at least 85% homologous andmore preferably 90% homologous and most preferably 95% homologous with anucleotide sequence set forth in a SEQ ID Nos. of the invention.Polypeptides which are encoded by a nucleic acid that is at least about98-99% homologous with the sequence of a SEQ ID No. of the invention arealso within the scope of the invention.

[0196] In a preferred embodiment, an Antigen protein of the presentinvention is a mammalian Antigen protein. In a particularly preferredembodiment an Antigen protein is set forth as a SEQ ID No. of theinvention. In particularly preferred embodiments, an Antigen protein hasan Antigen bioactivity. It will be understood that certainpost-translational modifications, e.g., phosphorylation and the like,can increase the apparent molecular weight of the Antigen proteinrelative to the unmodified polypeptide chain.

[0197] The invention also features protein isoforms encoded by splicevariants of the present invention. Such isoforms may have biologicalactivities identical to or different from those possessed by the Antigenproteins specified by a SEQ ID No. of the invention. Such isoforms mayarise, for example, by alternative splicing of one or more Antigen genetranscripts.

[0198] Antigen polypeptides preferably are capable of functioning aseither an agonist or antagonist of at least one biological activity of awild-type (“authentic”) Antigen protein of the appended sequencelisting. The term “evolutionarily related to”, with respect to aminoacid sequences of Antigen proteins, refers to both polypeptides havingamino acid sequences which have arisen naturally, and also to mutationalvariants of human Antigen polypeptides which are derived, for example,by combinatorial mutagenesis.

[0199] Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 20, 25, 50, 75 and 100, amino acids in length are withinthe scope of the present invention.

[0200] For example, isolated Antigen polypeptides can be encoded by allor a portion of a nucleic acid sequence shown in any of the sequencesshown in FIG. 8B or 9B or a SEQ ID No. of the invention. Isolatedpeptidyl portions of Antigen proteins can be obtained by screeningpeptides recombinantly produced from the corresponding fragment of thenucleic acid encoding such peptides. In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Forexample, an Antigen polypeptide of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of a wild-type(e.g., “authentic”) Antigen protein.

[0201] An Antigen polypeptide can be a membrane bound form or a solubleform. A preferred soluble Antigen polypeptide is a polypeptide whichdoes not contain a hydrophobic signal sequence domain. Such proteins canbe created by genetic engineering by methods known in the art. Thesolubility of a recombinant polypeptide may be increased by deletion ofhydrophobic domains, such as predicted transmembrane domains, of thewild type protein.

[0202] In general, polypeptides referred to herein as having an activity(e.g., are “bioactive”) of an antigen protein are defined aspolypeptides which include an amino acid sequence encoded by all or aportion of the nucleic acid sequences shown in one of the subject SEQ IDNos. and which mimic or antagonize all or a portion of thebiological/biochemical activities of a naturally occurring Antigenprotein. Examples of such biological activity include a region ofconserved structure.

[0203] Other biological activities of the subject Antigen proteins willbe reasonably apparent to those skilled in the art. According to thepresent invention, a polypeptide has biological activity if it is aspecific agonist or antagonist of a naturally-occurring form of anAntigen protein.

[0204] Assays for determining whether a compound, e.g, a protein, suchas an Antigen protein or variant thereof, has one or more of the abovebiological activities include those assays, well known in the art, whichare used for assessing Antigen agonist and Antigen antagonistactivities.

[0205] Other preferred proteins of the invention are those encoded bythe nucleic acids set forth in the section pertaining to nucleic acidsof the invention. In particular, the invention provides fusion proteins,e.g., Antigen-immunoglobulin fusion proteins. Such fusion proteins canprovide, e.g., enhanced stability and solubility of Antigen proteins andmay thus be useful in therapy. Fusion proteins can also be used toproduce an immunogenic fragment of an Antigen protein. For example, theVP6 capsid protein of rotavirus can be used as an immunologic carrierprotein for portions of the Antigen polypeptide, either in the monomericform or in the form of a viral particle. The nucleic acid sequencescorresponding to the portion of a subject Antigen protein to whichantibodies are to be raised can be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressingfusion proteins comprising Antigen epitopes as part of the virion. Ithas been demonstrated with the use of immunogenic fusion proteinsutilizing the Hepatitis B surface antigen fusion proteins thatrecombinant Hepatitis B virions can be utilized in this role as well.Similarly, chimeric constructs coding for fusion proteins containing aportion of an Antigen protein and the poliovirus capsid protein can becreated to enhance immunogenicity of the set of polypeptide antigens(see, for example, EP Publication No: 0259149; and Evans et al. (1989)Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger etal. (1992) J. Virol. 66:2).

[0206] The Multiple antigen peptide system for peptide-basedimmunization can also be utilized to generate an immunogen, wherein adesired portion of an Antigen polypeptide is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see, for example, Posnett et al. (1988) JBC 263:1719 andNardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants ofAntigen proteins can also be expressed and presented by bacterial cells.

[0207] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the Antigen polypeptides of the present invention. Forexample, Antigen polypeptides can be generated asglutathione-Stransferase (GST-fusion) proteins. Such GST-fusion proteinscan enable easy purification of the Antigen polypeptide, as for exampleby the use of glutathione-derivatized matrices (see, for example,Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: JohnWiley & Sons, 1991)). Additionally, fusion of Antigen polypeptides tosmall epitope tags, such as the FLAG or hemagluttinin tag sequences, canbe used to simplify immunological purification of the resultingrecombinant polypeptide or to facilitate immunological detection in acell or tissue sample. Fusion to the green fluorescent protein, andrecombinant versions thereof which are known in the art and availablecommercially, may further be used to localize Antigen polypeptideswithin living cells and tissue.

[0208] The present invention further pertains to methods of producingthe subject Antigen polypeptides. For example, a host cell transfectedwith a nucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant Antigenpolypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant Antigen polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

[0209] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject Antigen polypeptides which function in a limited capacity as oneof either an Antigen agonist (mimetic) or an Antigen antagonist, inorder to promote or inhibit only a subset of the biological activitiesof the naturally-occurring form of the protein. Thus, specificbiological effects can be elicited by treatment with a homolog oflimited function, and with fewer side effects relative to treatment withagonists or antagonists which are directed to all of the biologicalactivities of naturally occurring forms of Antigen proteins.

[0210] Homologs of each of the subject Antigen proteins can be generatedby mutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the Antigen polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an Antigen receptor.

[0211] The recombinant Antigen polypeptides of the present inventionalso include homologs of the wildtype Antigen proteins, such as versionsof those protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter ubiquitination or other enzymatictargeting associated with the protein.

[0212] Antigen polypeptides may also be chemically modified to createAntigen derivatives by forming covalent or aggregate conjugates withother chemical moieties, such as glycosyl groups, lipids, phosphate,acetyl groups and the like. Covalent derivatives of Antigen proteins canbe prepared by linking the chemical moieties to functional groups onamino acid sidechains of the protein or at the N-terminus or at theC-terminus of the polypeptide.

[0213] Modification of the structure of the subject Antigen polypeptidescan be for such purposes as enhancing therapeutic or prophylacticefficacy, stability (e.g., ex vivo shelf life and resistance toproteolytic degradation), or post-translational modifications (e.g., toalter phosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the Antigen polypeptides describedin more detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition. Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

[0214] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional Antigen homolog (e.g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

[0215] This invention further contemplates a method for generating setsof combinatorial mutants of the subject Antigen proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g., homologs). The purpose of screening suchcombinatorial libraries is to generate, for example, novel Antigenhomologs which can act as either agonists or antagonist, oralternatively, possess novel activities all together. Thus,combinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein.

[0216] In one embodiment, the variegated Antigen libary of Antigenvariants is generated by combinatorial mutagenesis at the nucleic acidlevel, and is encoded by a variegated gene Antigen library. Forinstance, a mixture of synthetic oligonucleotides can be enzymaticallyligated into gene sequences such that the degenerate set of potentialAntigen sequences are expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of Antigen sequences therein.

[0217] There are many ways by which such libraries of potential Antigenhomologs can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then ligated intoan appropriate expression vector. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential Antigen sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc3^(rd) Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam:Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477. Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al. (1990)Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

[0218] Likewise, a library of coding sequence fragments can be providedfor an Antigen clone in order to generate a variegated population ofAntigen fragments for screening and subsequent selection of bioactivefragments. A variety of techniques are known in the art for generatingsuch 1, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of an Antigen coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule;(ii) denaturing the double stranded DNA; (iii) renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

[0219] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of Antigen homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting libraries of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate Antigen sequences created bycombinatorial mutagenesis techniques. Combinatorial mutagenesis has apotential to generate very large libraries of mutant proteins, e.g., inthe order of 1026 molecules. Combinatorial libraries of this size may betechnically challenging to screen even with high throughput screeningassays. To overcome this problem, a new technique has been developedrecently, recrusive ensemble mutagenesis (REM), which allows one toavoid the very high proportion of non-functional proteins in a randomlibrary and simply enhances the frequency of functional proteins, thusdecreasing the complexity required to achieve a useful sampling ofsequence space. REM is an algorithm which enhances the frequency offunctional mutants in a library when an appropriate selection orscreening method is employed (Arkin and Yourvan, 1992, PNAS USA89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving fromNature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co.,Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering6(3):327-331).

[0220] The invention also provides for reduction of the Antigen proteinsto generate mimetics, e.g., peptide or non-peptide agents, such as smallmolecules, which are able to disrupt binding of an Antigen polypeptideof the present invention with a molecule, e.g. target peptide. Thus,such mutagenic techniques as described above are also useful to map thedeterminants of the Antigen proteins which participate inprotein-protein interactions involved in, for example, binding of thesubject Antigen polypeptide to a target peptide. To illustrate, thecritical residues of a subject Antigen polypeptide which are involved inmolecular recognition of its receptor can be determined and used togenerate Antigen derived peptidomimetics or small molecules whichcompetitively inhibit binding of the authentic Antigen protein with thatmoiety. By employing, for example, scanning mutagenesis to map the aminoacid residues of the subject Antigen proteins which are involved inbinding other proteins, peptidomimetic compounds can be generated whichmimic those residues of the Antigen protein which facilitate theinteraction. Such mimetics may then be used to interfere with the normalfunction of an Antigen protein. For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9^(th)American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985),b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; andSato et al. (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al.(1986) Biochem Biophys Res Commun 134:71).

[0221] “Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence contains an aminoacid sequence of at least 3 to 5 amino acids, more preferably at least 8to 10 amino acids, and even more preferably at least 15 to 20 aminoacids, a polypeptide encoded by the nucleic acid sequences. Alsoencompassed are polypeptide sequences which are immunologicallyidentifiable with a polypeptide encoded by the sequence. Thus, anantigen “polypeptide,” “protein,” or “amino acid” sequence may have atleast 60% similarity, preferably at least about 75% similarity, morepreferably about 85% similarity, and most preferably about 95%similarity, to a polypeptide or amino acid sequence of an antigen. Thisamino acid sequence can be selected from the group consisting of thepolypeptide sequence shown in FIG. 8B or 9B.

[0222] A “recombinant polypeptide” or “recombinant protein” or“polypeptide produced by recombinant techniques,” which are usedinterchangeably herein, describes a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system.

[0223] The term “synthetic peptide” as used herein means a polymericform of amino acids of any length, which may be chemically synthesizedby methods well-known to the routineer. These synthetic peptides areuseful in various applications.

[0224] The term “polynucleotide” as used herein means a polymeric formof nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA,as well as, double- and single-stranded RNA. It also includesmodifications, such as methylation or capping, and unmodified forms ofthe polynucleotide. The terms “polynucleotide,” “oligomer,”“oligonucleotide,” and “oligo” are used interchangeably herein.

[0225] “A sequence corresponding to a cDNA” means that the sequencecontains a polynucleotide sequence that is identical to or complementaryto a sequence in the designated DNA. The degree (or “percent”) ofidentity or complementarity to the cDNA will be approximately 50% orgreater, will preferably be at least about 70% or greater, and morepreferably will be at least about 90%. The sequence that corresponds tothe identified cDNA will be at least about 50 nucleotides in length,will preferably be about 60 nucleotides in length, and more preferably,will be at least about 70 nucleotides in length. The correspondencebetween the gene or gene fragment of interest and the cDNA can bedetermined by methods known in the art, and include, for example, adirect comparison of the sequenced material with the cDNAs described, orhybridization and digestion with single strand nucleases, followed bysize determination of the digested fragments.

[0226] “Purified polynucleotide” refers to a polynucleotide of interestor fragment thereof which is essentially free, i.e., contains less thanabout 50%, preferably less than about 70%, and more preferably, lessthan about 90% of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

[0227] “Purified polypeptide” means a polypeptide of interest orfragment thereof which is essentially free, that is, contains less thanabout 50%, preferably less than about 70%, and more preferably, lessthan about 90% of cellular components with which the polypeptide ofinterest is naturally associated. Methods for purifying are known in theart.

[0228] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

[0229] “Polypeptide” and “protein” are used interchangeably herein andindicates a molecular chain of amino acids linked through covalentand/or noncovalent bonds. The terms do not refer to a specific length ofthe product. Thus, peptides, oligopeptides and proteins are includedwithin the definition of polypeptide. The terms include post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. In addition, proteinfragments, analogs, mutated or variant proteins, fusion proteins and thelike are included within the meaning of polypeptide.

[0230] A “fragment” of a specified polypeptide refers to an amino acidsequence which comprises at least about 3-5 amino acids, more preferablyat least about 8-10 amino acids, and even more preferably at least about15-20 amino acids, derived from the specified polypeptide.

[0231] “Recombinant host cells,” “host cells,” “cells,” “cell lines,”“cell cultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

[0232] As used herein “replicon” means any genetic element, such as aplasmid, a chromosome or a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell.

[0233] A “vector” is a replicon in which another polynucleotide segmentis attached, such as to bring about the replication and/or expression ofthe attached segment.

[0234] The term “control sequence” refers to polynucleotide sequenceswhich are necessary to effect the expression of coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism. In prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site and terminators; ineukaryotes, such control sequences generally include promoters,terminators and, in some instances, enhancers. The term “controlsequence” thus is intended to include at a minimum all components whosepresence is necessary for expression, and also may include additionalcomponents whose presence is advantageous, for example, leadersequences.

[0235] “Operably linked” refers to a situation wherein the componentsdescribed are in a relationship permitting them to function in theirintended manner. Thus, for example, a control sequence “operably linked”to a coding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

[0236] The term “open reading frame” or “ORF” refers to a region of apolynucleotide sequence which encodes a polypeptide; this region mayrepresent a portion of a coding sequence or a total coding sequence.

[0237] A “coding sequence” is a polynucleotide sequence which istranscribed into mRNA and translated into a polypeptide when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to, mRNA, cDNA, and recombinantpolynucleotide sequences.

[0238] The term “immunologically identifiable with/as” refers to thepresence of epitope(s) and polypeptide(s) which also are present in andare unique to the designated polypeptide(s). Immunological identity maybe determined by antibody binding and/or competition in binding. Thesetechniques are known to the routineer and also are described herein. Theuniqueness of an epitope also can be determined by computer searches ofknown data banks, such as GenBank, for the polynucleotide sequenceswhich encode the epitope, and by amino acid sequence comparisons withother known proteins.

[0239] As used herein, “epitope” means an antigenic determinant of apolypeptide. Conceivably, an epitope can comprise three amino acids in aspatial conformation which is unique to the epitope. Generally, anepitope consists of at least five such amino acids, and more usually, itconsists of at least eight to ten amino acids. Methods of examiningspatial conformation are known in the art and include, for example,x-ray crystallography and two-dimensional nuclear magnetic resonance.

[0240] A “conformational epitope” is an epitope that is comprised ofspecific juxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

[0241] A polypeptide is “immunologically reactive” with an antibody whenit binds to an antibody due to antibody recognition of a specificepitope contained within the polypeptide. Immunological reactivity maybe determined by antibody binding, more particularly by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

[0242] As used herein, the term “immunogenic polypeptide containing anepitope of interest” means naturally occurring polypeptides of interestor fragments thereof, as well as polypeptides prepared by other means,for example, by chemical synthesis or the expression of the polypeptidein a recombinant organism.

[0243] The term “transformation” refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction or f-mating areincluded. The exogenous polynucleotide may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may beintegrated into the host genome.

[0244] “Treatment” refers to prophylaxis and/or therapy.

[0245] The term “individual” as used herein refers to vertebrates,particularly members of the mammalian species and includes but is notlimited to domestic animals, sports animals, primates and humans; moreparticularly the term refers to humans.

[0246] The term “sense strand” or “plus strand” (or “+”) as used hereindenotes a nucleic acid that contains the sequence that encodes thepolypeptide. The term “antisense strand” or “minus strand” (or “−”)denotes a nucleic acid that contains a sequence that is complementary tothat of the “plus” strand.

[0247] The term “test sample” refers to a component of an individual'sbody which is the source of the analyte (such as, antibodies of interestor antigens of interest). These components are well known in the art.These test samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitorurinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants; fixed tissue specimens; and fixed cell specimens.

[0248] “Purified product” refers to a preparation of the product whichhas been isolated from the cellular constituents with which the productis normally associated, and from other types of cells which may bepresent in the sample of interest.

[0249] “PNA” denotes a “peptide nucleic acid analog” which may beutilized in a procedure such as an assay described herein to determinethe presence of a target. “MA” denotes a “morpholino analog” which maybe utilized in a procedure such as an assay described herein todetermine the presence of a target. See, for example, U.S. Pat. No.5,378,841, which is incorporated herein by reference. PNAs are neutrallycharged moieties which can be directed against RNA targets or DNA. PNAprobes used in assays in place of, for example, the DNA probes of thepresent invention, offer advantages not achievable when DNA probes areused. These advantages include manufacturability, large scale labeling,reproducibility, stability, insensitivity to changes in ionic strengthand resistance to enzymatic degradation which is present in methodsutilizing DNA or RNA. These PNAs can be labeled with such signalgenerating compounds as fluorescein, radionucleotides, chemiluminescentcompounds, and the like. PNAs or other nucleic acid analogs such as MAsthus can be used in assay methods in place of DNA or RNA. Althoughassays are described herein utilizing DNA probes, it is within the scopeof the routineer that PNAs or MAs can be substituted for RNA or DNA withappropriate changes if and as needed in assay reagents.

[0250] “Analyte,” as used herein, is the substance to be detected whichmay be present in the test sample. The analyte can be any substance forwhich there exists a naturally occurring specific binding member (suchas, an antibody), or for which a specific binding member can beprepared. Thus, an analyte is a substance that can bind to one or morespecific binding members in an assay. “Analyte” also includes anyantigenic substances, haptens, antibodies, and combinations thereof. Asa member of a specific binding pair, the analyte can be detected bymeans of naturally occurring specific binding partners (pairs) such asthe use of intrinsic factor protein as a member of a specific bindingpair for the determination of Vitamin B12, the use of folate-bindingprotein to determine folic acid, or the use of a lectin as a member of aspecific binding pair for the determination of a carbohydrate. Theanalyte can include a protein, a peptide, an amino acid, a nucleotidetarget, and the like.

[0251] “Inflammation” or “inflammatory disease,” as used herein, referto infiltration of activated lymphocytes such as neutrophils,eosinophils, macrophages, T cells and B-cells, into a host tissue thatresults in damage to the host organism. Examples of inflammatory diseaseinclude but are not limited to conditions such as inflammatory boweldisease, sepsis, and rheumatoid arthritis.

[0252] An “Expressed Sequence Tag” or “EST” refers to the partialsequence of a cDNA insert which has been made by reverse transcriptionof mRNA extracted from a tissue, followed by insertion into a vector.

[0253] A “transcript image” refers to a table or list giving thequantitative distribution of ESTs in a library and represents the genesactive in the tissue from which the library was made.

[0254] The present invention provides assays which utilize specificbinding members. A “specific binding member,” as used herein, is amember of a specific binding pair. That is, two different moleculeswhere one of the molecules through chemical or physical meansspecifically binds to the second molecule. Therefore, in addition toantigen and antibody specific binding pairs of common immunoassays,other specific binding pairs can include biotin and avidin,carbohydrates and lectins, complementary nucleotide sequences, effectorand receptor molecules, cofactors and enzymes, enzyme inhibitors andenzymes, and the like. Furthermore, specific binding pairs can includemembers that are analogs of the original specific binding members, forexample, an analyte-analog. Immunoreactive specific binding membersinclude antigens, antigen fragments, antibodies and antibody fragments,both monoclonal and polyclonal, and complexes thereof, including thoseformed by recombinant DNA molecules.

[0255] The term “hapten,” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

[0256] A “capture reagent,” as used herein, refers to an unlabeledspecific binding member which is specific either for the analyte as in asandwich assay, for the indicator reagent or analyte as in a competitiveassay, or for an ancillary specific binding member, which itself isspecific for the analyte, as in an indirect assay. The capture reagentcan be directly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

[0257] The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means, conjugated (“attached”) to aspecific binding member. “Specific binding member” as used herein meansa member of a specific binding pair. That is, two different moleculeswhere one of the molecules through chemical or physical meansspecifically binds to the second molecule. In addition to being anantibody member of a specific binding pair, the indicator reagent alsocan be a member of any specific binding pair, including eitherhapten-anti-hapten systems such as biotin or anti-biotin, avidin orbiotin, a carbohydrate or a lectin, a complementary nucleotide sequence,an effector or a receptor molecule, an enzyme cofactor and an enzyme, anenzyme inhibitor or an enzyme, and the like. An immunoreactive specificbinding member can be an antibody, an antigen, or an antibody/antigencomplex that is capable of binding either to polypeptide of interest asin a sandwich assay, to the capture reagent as in a competitive assay,or to the ancillary specific binding member as in an indirect assay.When describing probes and probe assays, the term “reporter molecule”may be used. A reporter molecule comprises a signal generating compoundas described hereinabove conjugated to a specific binding member of aspecific binding pair, such as carbazol or adamantane.

[0258] 4.6. Stem Cell Preparation and Manipulation

[0259] Methods for isolating and manipulating bone marrow cells,including hematopoietic stem cells, from a bone marrow graft donor areknown in the art. For example, U.S. Pat. Nos. 4,965,204, 5,035,994,5,081,030, 5,130,144, 5,137,809, 6,068,836 and 6,200,606, the contentsof which patents are hereby incorporated by reference, describe methodsfor obtaining and manipulating bone marrow stem cells from a mammalianbone marrow donor. One of the most useful differentiation antigens forisolating human hematopoietic systems is the cell surface antigen knownas CD34. CD34 is expressed by about 1% to 5% of normal human adultmarrow cells in a developmentally, stage-specific manner (C I Civin etal., “Antigenic analysis of hematopoiesis. A hematopoietic progenitorcell surface antigen defined by a monoclonal antibody raised againstKG-1a cells” J. Immunol., 133, 157-165, 1984). CD34+ cells are a mixtureof immature blastic cells and a small percentage of mature,lineage-committed cells of the myeloid, erythroid and lymphoid series.Perhaps 1% of CD34+ cells are true HSC with the remaining number beingcommitted to a particular lineage. Results in humans have demonstratedthat CD34+ cells isolated from peripheral blood or marrow canreconstitute the entire hematopoietic system for a lifetime. Therefore,CD34 is a marker for HSC and hematopoietic progenitor cells.

[0260] For example, selective cytapheresis can be used to produce a cellsuspension from human bone marrow or blood containing pluripotentlymphohematopoeitic stem cells. For example, marrow can be harvestedfrom a donor (the patient in the case of an autologous transplant; adonor in the case of an allogenic transplant) by any appropriate means.The marrow can be processed as desired, depending mainly upon the useintended for the recovered cells. The suspension of marrow cells isallowed to physically contact, for example, a solid phase-linkedmonoclonal antibody that recognizes an antigen on the desired cells. Thesolid phase-linking can comprise, for instance, adsorbing the antibodiesto a plastic, nitrocellulose or other surface. The antibodies can alsobe adsorbed on to the walls of the large pores (sufficiently large topermit flow-through of cells) of a hollow fiber membrane. Alternatively,the antibodies can be covalently linked to a surface or bead, such asPharmacia Sepharose 6 MB macrobeads.RTM. The exact conditions andduration of incubation for the solid phase-linked antibodies with themarrow cell suspension will depend upon several factors specific to thesystem employed. The selection of appropriate conditions, however, iswell within the skill of the art.

[0261] The unbound cells are then eluted or washed away with physiologicbuffer after allowing sufficient time for the stem cells to be bound.The unbound marrow cells can be recovered and used for other purposes ordiscarded after appropriate testing has been done to ensure that thedesired separation had been achieved. The bound cells are then separatedfrom the solid phase by any appropriate method, depending mainly uponthe nature of the solid phase and the antibody. For example, bound cellscan be eluted from a plastic petrie dish by vigorous agitation.Alternatively, bound cells can be eluted by enzymatically “nicking” ordigesting a enzyme-sensitive “spacer” sequence between the solid phaseand the antibody. Spacers bound to agarose beads are commerciallyavailable from, for example, Pharmacia.

[0262] The eluted, enriched fraction of cells may then be washed with abuffer by centrifugation and either cryopreserved in a viable state forlater use according to conventional technology or immediately infusedintravenously into the transplant recipient.

[0263] In a particularly preferred embodiment, stem cells can berecovered directly from blood using essentially the above methodology.For example, blood can be withdrawn directly from the circulatory systemof a donor and percolated continuously through a device (e.g., a column)containing the solid phase-linked monoclonal antibody to stem cells andthe stem cell-depleted blood can be returned immediately to the donor'scirculatory system using, for example, a conventional hemapheresismachine. When a sufficient volume of blood has been processed to allowthe desired number of stem cells to bind to the column, the patient isdisconnected. Such a method is extremely desirable because it allowsrare peripheral blood stem cells to be harvested from a very largevolume of blood, sparing the donor the expense and pain of harvestingbone marrow and the associated risks of anesthesia, analgesia, bloodtransfusion, and infection. The duration of aplasia for the transplantrecipient following the marrow transplant can also be shortened since,theoretically, unlimited numbers of blood stem cells could be collectedwithout significant risk to the donor.

[0264] The above methods of treating marrow or blood cell suspensionsproduce a suspension of human cells that contains pluripotentlympho-hematopoietic stem cells, but substantially free of maturelymphoid and myeloid cells. The cell suspension also containssubstantially only cells that express the My-10 antigen and can restorethe production of lymphoid and hematopoietic cells to a human patientthat has lost the ability to produce such cells because of, for example,radiation treatment. By definition, a cell population that can restorethe production of hematopoietic and lymphoid cells contains pluripotentlympho-hematopoietic stem cells.

[0265] The above cell populations containing human pluripotentlympho-hematopoetic stem cells can be used in therapeutic methods suchas stem cell transplantation as well as others that are readily apparentto those of skill in the art. For example, such cell populations can beadministered directly by I.V. to a patient requiring a bone marrowtransplant in an amount sufficient to reconstitute the patient'shematopoietic and immune system. Precise, effective quantities can bereadily determined by those skilled in the art and will depend, ofcourse, upon the exact condition being treated by the therapy. In manyapplications, however, an amount containing approximately the samenumber of stem cells found in one-half to one liter of aspirated marrowshould be adequate.

[0266] 4.7. Dendritic Cell Preparation and Manipulation

[0267] Methods for isolating and manipulating dendritic cells, includingdendritic antigen presenting cells from a bone marrow graft sample orfrom peripheral blood of a donor animal are known in the art. Forexample, U.S. Pat. Nos. 6,165,785 and 6,194,204, the contents of whichpatents are hereby incorporated by reference, describe methods forobtaining and manipulating dendritic (antigen-presenting) cells from amammalian bone marrow donor.

[0268] One exemplary method for the enrichment of dendritic cells fromthe peripheral blood of a mammal utilizes the following steps. Themononuclear cells are separated from the peripheral blood. Themononuclear cells are separated into a first cell population havingsubstantially lymphocytes and a second cell population havingsubstantially myeloid cells. The myeloid cells are separated into athird cell population having substantially monocytes and a fourth cellpopulation having substantially dendritic cells.

[0269] First, the mononuclear cells are separated from the peripheralblood. These mononuclear cells are separated into a first cellpopulation having substantially lymphocytes and a second cell populationhaving substantially myeloid cells. These myeloid cells are separatedinto a third cell population having substantially monocytes and a fourthcell population having substantially dendritic cells.

[0270] By peripheral blood is meant blood found in the circulationvasculature. The peripheral blood can be obtained from any mammal. Bymammal is meant human as well as non-human mammal. Preferred non-humanmammals is a mouse or a pig. Preferably, the peripheral blood isobtained from the same source from which the donor stem cell graft, e.g.the hematopoietic stem cell graft, is obtained. The mononuclear cellscan be separated from the peripheral blood by any method known to thoseskilled in the art. Preferably, the method used does not affect cellfunction or viability. A preferred method is the use of centrifugation,preferably density gradient centrifugation, preferably discontinuousdensity gradient centrifugation. An alternative is the use of specificmonoclonal antibodies.

[0271] The mononuclear cells are separated into a first cell populationhaving substantially lymphocytes and a second cell population havingsubstantially myeloid cells. Lymphocytes are meant to include, e.g., Tcells, NK cells, B cells and mixtures thereof. By a cell populationhaving substantially lymphocytes is meant that the cell population hasgreater than about 20% lymphocytes, preferably greater than about 40%lymphocytes, more preferably greater than about 60% lymphocytes, morepreferably yet greater than about 80% lymphocytes, more preferably yetgreater than about 90% lymphocytes, more preferably yet greater thanabout 95% lymphocytes, more preferably yet greater than about 98%lymphocytes, and most preferably greater than about 99% lymphocytes.Myeloid cells are meant to include monocytes and dendritic cells.Monocytes are also meant to include macrophages. It is known thatmonocytes circulate in the peripheral blood, and when they migrate tothe tissue, they are called macrophages. This lineage of cells arecommonly called monocyte/macrophage lineage. Myeloid cells are generallyCD14.sup.+, CD33.sup.+ and CD13.sup.+. By a cell population havingsubstantially myeloid cells is meant that the cell population hasgreater than about 20% myeloid cells, preferably greater than about 40%myeloid cells, more preferably greater than about 60% myeloid cells,more preferably yet greater than about 80% myeloid cells, morepreferably yet greater than about 90% myeloid cells, more preferably yetgreater than about 95% myeloid cells, more preferably yet greater thanabout 98% myeloid cells, and most preferably greater than about 99%myeloid cells.

[0272] In certain embodiments, the separation of the mononuclear cellsinto a first cell population having substantially lymphocytes and asecond cell population having substantially myeloid cells comprisescontacting the mononuclear cells with antibodies against the lymphocytesso as to form an antibody-lymphocyte complex, and selectively separatingthe antibody-lymphocyte complex from the myeloid cells. One or more thanone type of antibody can be used. In certain embodiments, the contactingand the selectively separating steps are repeated. These steps can berepeated using the same type of antibody or antibodies against thelymphocytes, or they can be repeated using a different type of antibodyor antibodies against the lymphocytes.

[0273] Both polyclonal and monoclonal antibodies can be used in thisinvention. Preferably, monoclonal antibodies are used. Antibodiesagainst the lymphocytes include, e.g., T cell antibodies, NK cellantibodies, B cell antibodies, or mixtures thereof. Preferably, mixturesof the antibodies are used. The antibodies used are directed against oneor more antigens which are expressed by one or more of the lymphocytes.

[0274] Preferably, the T cell antibodies are anti-CD3 antibodies. All Tcells express the CD3 surface molecule. CD3 is described in Barclay etal., The Leukocyte Antigen Facts Book, Academic Press Limited (1993),pp. 106-109. Anti-CD3 antibodies can be obtained from Becton DickinsonImmunocytometry Systems, San Jose, Calif. or Coulter Corp., Miami, Fla.Other T cell antibodies that can be used include, e.g., anti-CD8antibodies. CD8 is described in Barclay et al., The Leukocyte AntigenFacts Book, Academic Press Limited (1993), pp. 118-119. Anti-CD8antibodies can be obtained from Becton Dickinson Immunocytometry Systemsor Coulter Corp. Not all T cells express CD8. CD8 is expressed byroughly 40% of the T-lymphocyte population. Therefore, using, e.g.,anti-CD8 antibodies will generally not result in the separation of theentire T cell population from the myeloid cells. There are, however,certain situations in which it might be desirable to use anti-CD8antibodies. For example, CD.sup.8+T lymphocytes represent a cytotoxicT-lymphocyte population. This population selectively targets and killscells which were exposed to pathogen-specific antigens used in theproduction of pathogen-specific cytotoxic T cell lysis (intracellularpathogens).

[0275] Preferably, the NK cell antibodies are anti-CD16/56. CD 16/56refers to CD16 and CD56; they are not the same antigen, but are bothexpressed by NK cells. (CD8.sup.+T lymphocytes also express CD 16).Anti-CD 16/56 antibodies can be obtained from Becton DickinsonImmunocytometry Systems or Coulter Corp. In certain embodiments, the NKcell antibodies can be anti-CD8. Not all NK cells express CD8, andtherefore using anti-CD8 antibodies will not result in the separation ofthe entire NK cell population from the myeloid cells.

[0276] Preferably, the B cell antibodies are anti-CD19 or anti-CD20antibodies. CD19 and CD20 are expressed by resting and activated Blymphocytes. CD19 and CD20 are described in Barclay et al., TheLeukocyte Antigen Facts Book, Academic Press Limited (1993), pp. 142-143and 144-145, respectively. Anti-CD19 and anti-CD20 antibodies can beobtained from Becton Dickinson Immunocytometry Systems or Coulter Corp.

[0277] The antibody-lymphocyte complex that is formed is selectivelyseparated from the myeloid cells. In certain embodiments, thisseparation comprises contacting the antibody-lymphocyte complex and themyeloid cells with a matrix such that the antibody-lymphocyte complex issubstantially retained by the matrix and the myeloid cells aresubstantially not retained by the matrix. Any matrix which performs sucha separation can be used.

[0278] A matrix which is particularly useful is a mesh of steel woolwhich is inserted into a plastic column and placed in a magnetic field.A cell magnetic bead complex passes into the matrix and remains in thematrix as long as the column stays within the magnetic field. Examplesof matrices include depletion columns type BS, type CS, type D RS+, andMS+ used for Mini Mags separator. (All these columns can be obtainedfrom Miltenyi Biotec, Auburn, Calif.) Preferably, the matrix is providedin a column, though the matrix can be provided in any other way known tothose skilled in the art, e.g., in a gel, on a filter, on a plate, onfilm or on paper.

[0279] By the complex being substantially retained by the matrix ismeant that greater than about 20% of the complex is retained, preferablygreater than about 40% is retained, more preferably greater than about60% is retained, more preferably yet greater than about 80% is retained,more preferably yet greater than about 90% is retained, more preferablyyet greater than about 95% is retained, and most preferably greater thanabout 98% is retained. By the myeloid cells being substantially notretained by the matrix is meant that greater than about 20% of themyeloid cells are not retained, preferably greater than about 40% arenot retained, more preferably greater than about 60% are not retained,more preferably yet greater than about 80% are not retained, morepreferably yet greater than about 90% are not retained, more preferablyyet greater than about 95% are not retained, and most preferably greaterthan about 98% are not retained.

[0280] In preferred embodiments, the antibody-lymphocyte complex furthercomprises magnetic beads. Preferably, the magnetic beads aresuperparamagnetic microparticles, though any type of magnetic bead canbe used. The magnetic beads can be attached, e.g., to the antibody or tothe lymphocyte or to both. Preferably, the magnetic beads are attachedto the antibody. Such attached antibodies can be obtained, e.g., fromMiltenyi Biotec, Auburn, Calif. (as MACS superparamagnetic microbeadsconjugated with monoclonal antibodies), or from Dynal Corp., LakeSuccess, N.Y. (as detachable or non-detachable large magnetic beads).See also Miltenyi et al., Cytometry 11:231-238 (1990). Preferably, thelarge magnetic beads (obtainable from Dynal Corp.), are used for theremoval of lymphocytes. Preferably, the smaller beads (obtainable fromMiltenyi Biotec), are used for the enrichment of the dendritic cellsdescribed below. The magnetic beads can be attached prior to theformation of the antibody-lymphocyte complex, or subsequent to theformation of the complex. Preferably, the magnetic beads are attachedprior to formation of the complex.

[0281] In embodiments in which the antibody-lymphocyte complex hasmagnetic beads, separation of such a complex from the myeloid cellspreferably comprises contacting the myeloid cells and the complex with amagnetic matrix such that the antibody-lymphocyte complex having themagnetic beads is substantially retained by the magnetic matrix and themyeloid cells are substantially not retained by the magnetic matrix. Anexample of a magnetic matrix is magnetized steel wool. Steel wool can beobtained from Miltenyi Biotec. The steel wool can be magnetized by,e.g., introducing it into a magnetic field, e.g., 0.6 Tesla, thoughother strength magnetic fields can also be used as known to thoseskilled in the art. The magnetic field can be produced, e.g., with acommercial electromagnet.

[0282] In certain embodiments, the antibodies to the T cells, NK cellsand B cells are all contacted with the mononuclear cells prior toselectively separating the resulting antibody-lymphocyte complexes fromthe myeloid cells. In other embodiments, antibodies to only one type oflymphocyte cell are added (e.g., T cells), and the resultingantibody-lymphocyte complex is separated from the remaining cells.Antibodies to one of the remaining types of lymphocytes (e.g., NK cells)are then added to the remaining cells from above, and the resultingantibody-lymphocyte complex is separated from these remaining cells.Finally, antibodies to the remaining type of lymphocyte (e.g., B cells)are then added to this second batch of remaining cells, and theresulting antibody-lymphocyte complex is separated from these remainingcells (predominantly the myeloid cells). Preferably, all of theantibodies are added prior to selective separation.

[0283] The invention also includes embodiments in which separation ofthe mononuclear cells into a first cell population having substantiallylymphocytes and a second cell population having substantially myeloidcells, comprises centrifugation. The centrifugation can be, e.g.,density gradient centrifugation. For example, metrizamide 14.5%(obtained from Sigma Chemical Co., St. Louis, Mo.) or Monocyte I step(which is a pre-made discontinuous gradient which separates lymphocytesfrom myeloid cells, obtained from Accurate Chemical and ScientificCorp., Westbury, N.Y.), can be used. Centrifugation procedures are mostuseful if there are initially a large number of PBMCs, e.g., about10.sup.9.

[0284] In certain embodiments, the separation of the mononuclear cellsinto a third cell population having substantially monocytes and a fourthcell population having substantially dendritic cells comprisescontacting the myeloid cells with antibodies against the dendritic cellsso as to form an antibody-dendritic cell complex, and selectivelyseparating the antibody-dendritic cell complex from the monocytes. Incertain embodiments, the contacting and the selectively separating stepsare repeated. These steps can be repeated using the same type ofantibody or antibodies against the dendritic cells, or they can berepeated using a different type of antibody or antibodies against thedendritic cells.

[0285] Preferably, monoclonal antibodies are used. The antibodies usedare directed against one or more antigens which are expressed by thedendritic cells. Preferably, the antibodies are anti-CD2 antibodies,anti-CD5 antibodies, or mixtures thereof. Most preferably, anti-CD2antibodies are used because they stain greater than 95% of the dendriticcells and do not modulate down in culture. Mixtures of the antibodiescan also be used. CD2 and CD5 are described in Barclay et al., TheLeukocyte Antigen Facts Book, Academic Press Limited (1993), pp. 104-105and 112-113, respectively. Anti-CD2 antibodies can be obtained fromCoulter Corp. Anti-CD5 antibodies can be obtained from Becton DickinsonImmunocytometry Systems or Coulter Corp.

[0286] The CD2 antigen is a 50 kD molecular weight glycoprotein that wasinitially identified on T cells and NK cells and has now been shown inthis invention to be expressed by circulating dendritic cells.Antibodies to this surface antigen react strongly with resting T cells.The CD2 surface antigen is divided into three regions reflecting theirfunctional relationship. The first region, T11.sub. 1, is responsiblefor adhesion with the LFA-3 molecule and sheep erythrocyte binding. Thefirst antibody that was produced to this region is called T11.sub.1 andits clone designation is 3PTH29. The second region, T11.sub.2, is anarea on the CD2 antigen which does not interact with the binding domainbut has been demonstrated to play a role in T cell activation inconjunction with a second antibody. The first antibody that was producedto this region is called T11.sub.2 and its clone designation is1OLD24C1. Other T11.sub.2 clones are UMCD2/1E7E8, 0275, 9.6 and 7E10.The crosslinking of the Ti 1.sub.2 region with monoclonal antibodiesinduces unfolding of the CD2 antigen and exposure of a cryptic epitope.This cryptic epitope represents a third region, T11.sub.3 or CD2R, andis expressed by activated T cells and cell-lines but only after exposureto T11.sub.2 monoclonal antibodies (or others with similar traits),which induces a conformational change in structure of the CD2 antigen.The first antibody to this region was T11.sub.3 and its clone name is 1mono2A6. Other T11.sub.3 clones are VIT13, G144 and L304. In preferredembodiments, T11.sub.2 or T11.sub.2 plus T11.sub.3 antibodies are used.

[0287] In certain embodiments, prior to contacting the myeloid cellswith antibodies, the myeloid cells are cultured, preferably for about 12hours to about 36 hours, in about 5% to about 10% pooled mammal specificserum. For example, pooled human serum is used if the isolation is fromhuman peripheral blood, and pooled pig serum is used if the isolation isfrom pig peripheral blood. After such culturing, antibodies, preferablyanti-CD83 antibodies, can be used so as to form an antibody-dendriticcell complex. (CD83 is described in Zhou et al., J. Immunol. 154:3821-3835 (1995); Crawford et al., Blood 80(10) Suppl. 1:192a (1992)).Anti-CD83 antibodies can be isolated as described in Zhou et al., J.Immunol. 149:735 (1992). The dendritic cells that are isolated in thisembodiment can be phenotypically CD14.sup.-.

[0288] The antibody-dendritic cell complex that is formed, e.g., as aresult of using any of the antibodies described above, is selectivelyseparated from the monocytes. In certain embodiments, the separationcomprises contacting the antibody-dendritic cell complex and themonocytes with a matrix such that the antibody-dendritic cell complex issubstantially retained by the matrix and the monocytes are substantiallynot retained by the matrix. Preferably, the retained antibody-dendriticcell complex is then eluted from the matrix.

[0289] In preferred embodiments, the antibody-dendritic cell complexfurther comprises magnetic beads, as described above. In suchembodiments, separation of the antibody-dendritic complex from themonocytes preferably comprises contacting the monocytes andantibody-dendritic cell complex having the magnetic beads with amagnetic matrix such that the antibody-dendritic cell complex having themagnetic beads is substantially retained by the magnetic matrix and themonocytes are substantially not retained by the magnetic matrix.Preferably, the retained antibody-dendritic cell complex is then elutedfrom the matrix. The complex can be eluted, e.g., by demagnetizing thematrix, e.g., by removing the matrix from the magnetic field.

[0290] Preferably, the dendritic cells in the fourth cell population aregreater than about 60% pure, more preferably greater than about 70%pure, more preferably yet greater than about 80% pure, more preferablyyet greater than about 90% pure, more preferably yet greater than about95% pure, more preferably yet greater than about 98% pure, and mostpreferably greater than about 99% pure. In certain embodiments, thedendritic cells in the fourth cell population are substantiallyunactivated. In certain embodiments, the above method further comprisesthe step of activating the dendritic cells in the fourth cellpopulation, comprising culturing the dendritic cells with T11.sub.3antibodies or LFA-3 ligand.

[0291] Preferably, the monocytes in the third cell population aregreater than about 70% pure, more preferably greater than about 80%pure, more preferably yet greater than about 90% pure, more preferablyyet greater than about 95% pure, more preferably yet greater than about98% pure, and most preferably greater than about 99% pure. Preferably,the monocytes in the third cell population are substantiallyunactivated. An advantage of the present invention is that it canproduce monocytes which are unactivated. Other monocyte isolationprocedures which use plastic adherence are known to rapidly inducemonocyte activation. See Triglia et al., Blood 65(4):921-928 (1985).

[0292] The invention also includes a method for the enrichment ofdendritic cells from the peripheral blood of a mammal comprisingselecting cells from the peripheral blood which do not express antigensCD3, CD16/56 and CD19 or CD20, and which do express antigen CD2, CD5,CD83, or mixtures thereof. Preferably, cells are selected which alsoexpress antigen CD14. In certain embodiments, cells are selected whichdo not express antigen CD14.

[0293] The invention also includes a method for the enrichment ofdendritic cells from tissue of a mammal. Tissue having mononuclear cellsfrom a mammal is provided. The mononuclear cells are separated from thetissue. The mononuclear cells are separated into a first cell populationhaving substantially lymphocytes and a second cell population havingsubstantially myeloid cells. The myeloid cells are separated into athird cell population having substantially monocytes and a fourth cellpopulation having substantially dendritic cells. The tissue can be fromany part of the body of the mammal that has dendritic cells, e.g., skinor lymph nodes.

5. EXAMPLES

[0294] The examples below provide guidance to the skilled artisan inapplying the methods and compositions of the invention for modulation ofsystemic immune responses in a host organism by transplantation ofhematopoietic stem cells transduced with genes encoding antigens andantigen presenting cell regulatory molecules. The approach takesadvantage of bone marrow transplantation and hematopoietic stem celltransplantation which is currently routinely utilized clinically. Theinvention applies the capacity of modern vectors to transducehematopoietic stem cells or bone marrow cells ex vivo prior to theirtransplant. This procedures utilized are in keeping with standard bonemarrow manipulations that are currently utilized routinely. Anadditional element to this approach involves the infusion of eitherautologous or allogeneic mature lymphocytes subsequent to the bonemarrow transplant. Again, this approach is routinely performedclinically. The following examples provide guidance in establishing apopulation of antigen expressing cells in a host organism usingappropriate vectors such as those described as well as those which maybe adapted from these exemplary vectors. Accordingly, these examplesprovide guidance in developing various applications of the inventionincluding DNA vaccines directed against pathogens and tumor antigens aswell as in the development of treatments for autoimmune disease and forthe establishment of antigen tolerance.

Example 1 Approach to Efficient Recombinant Antigen Expression

[0295] Effective induction of systemic anti-tumor immunity requiresoptimal tumor antigen presentation by major histocompatibility complex(MHC) molecules simultaneously with appropriate costimulatory signals.Pardoll, D. M. (1998), Nat. Med 4:525; Herlyn, D. and Birebent, B.(1999), Ann. Med. 31:66; Banchereau, J. and Steirnan, R. M. (1998),Nature 392:245; and Hart, D. N. J. (1997), Blood 90:3245. Manytherapeutic and preventive tumor vaccination approaches have focused onengineering APCs, particularly DCs. While DC-based vaccines are nowpopular, recent studies using ex vivo antigen-loaded DC as cancervaccines have shown limited success. This appears to be due, in part, toinefficient numbers of ex vivo differentiated DCs that home to relevantlymphoid compartments, thereby limiting their access to T cells. DNAvaccines are relatively inefficient in transducing DCs in vivo, asdemonstrated by the paucity of transduced DCs found in lymph nodesdraining vaccine sites. These factors ultimately limit the stimulationof antigen-specific T cells by DC.

[0296] To overcome these hurdles, we will employ a different approach toallow efficient tumor antigen presentation by functional DCs matured invivo. Because ex vivo cultured and transduced HSC can home to bonemarrow (BM) and reconstitute the lymphohematopoietic system, we proposethat HSC transduced ex vivo with a specific tumor Ag gene can generatelarge numbers of DC that differentiate in vivo and then express thetumor Ag. This approach provides the potential to express relevantantigens in great numbers of DCs within the body rather than therelatively tiny number accessed by standard vaccines. We will presentpreliminary data that this approach indeed results in enhancedactivation of antigen-specific T cells transferred into the transplantedanimals. For this HSC-based approach, transgene expression is furthercontrolled by an improved lentiviral vector (LV) allowing selectiveexpression of the transgene in DCs derived from the transduced HSC.Together with the application of various specific promoters driving thetransgene, this HSC-based transduction approach also allows us toexamine the in vivo immunoregulatory functions of different DC subsets.Herlyn, D. and Birebent, B. (1999), Ann. Med. 31:66; Banchereau, J. andSteinman, R. M. (1998), Nature 392:245; and Hart, D. N. J. (1997), Blood90:3245. Importantly, the success of our approach does not rely on highefficiency transduction of the most primitive HSC. Generation ofAg-expressing DCs from transduced multipotent progenitors may besufficient to immunize the organism. Indeed, the high transductionefficiencies of engrafting human and mouse HSC that we demonstrate belowindicate that HSC transduction efficiency is unlikely to be therate-limiting step. We will compare these in vivo and ex vivodifferentiated, Ag-expressing DCs in their functional capacity to primeanticancer immune responses and ultimately to reverse immune toleranceto these tumor antigens, which represents the major barrier to effectivecancer immunotherapy.

Example 2 HSC Gene transduction by LV and Oncoretroviral Vectors (RV)

[0297] With improved methods for HSC culture and virus production, wecan now transduce human HSC, assayed as SCID-repopulating cells (SRC) inNOD/SCID mice, using RV or LV. These methods are described in: Cheng, L.et al. (1997), Gene Therapy 4:1013; Cheng, L. et al. (1998), Blood92:83; Novelli, E. et al. (1999), Human Gene Therapy 10:2927; Gao, Z. etal. (2000), Molecular Therapy 1:35S; Gao, Z. et al. (2001), Stem Cells,in press; and Cui, Y. et al. (2000), Exp. Hematology 27:62a, thecontents of which are incorporated by reference. Recently we have made adirect comparison of MSCV-based RV with HIV-based LV for SRC genetransduction. These (see Gao, Z. et al. (2000), Molecular Therapy 1:35S;and Gao, Z. et al. (2001), Stem Cells, in press) and other datademonstrate that LV require less ex vivo manipulations and are moreefficient to transduce human HSC than RV.

EXAMPLE 3 Stable and Targeted Transgene Expression Mediated by LV

[0298] It has generally been difficult to construct high-titer RV whosetransgenes are solely under the control of a non-viral (internal)promoter. Recently it was demonstrated that specific transgeneexpression controlled by an internally-built specific promoter is morereadily achieved using LV with self-inactivating modification; Cui, Y.et al. (1999), J Virology 73:6171; and Zufferey, R. et al. (1998), J.Virol. 72:9873; which disables the basal promoter function of the LV LTRpost integration (FIG. 1). Since this project depends in part ongenerating DC-specific gene expression, the superiority of LV to RV inthis regard further validates LV as the vector of choice. We began byusing a SIN vector (EF.GFP) in which transgene expression (GFP) iscontrolled by the promoter of a human housekeeping gene, EF1a (FIG. 2A).To further examine the feasibility of selective transgene expression inAPCs, we made the DR.GFP vector. In this LV, the human EF1a promoter wasreplaced by the human HLA-DR□ (MHC class II) promoter, whose gene isexpressed selectively in APCs and highly in mature, activated DCs. Insubsequent experiments, we evaluated the performance of SIN vectorsdriving the GFP reporter in DCs differentiated in vitro and in vivo fromtransduce human CD34(+) cells and mouse BM cells.

EXAMPLE 4 Preferential Transgene Expression of the DR. GFP Vector inHuman MHC II⁺ Cells Differentiated From L V-Transduced Human CD34⁺ cells

[0299] After 1-2 rounds of transduction with DR.GFP or EF.GFP LV,transduced human cord or adult blood CD34⁺ cells were cultured for 7days with GM-CSF/IL-4/TNFa under conditions favoring DC differentiation(FIG. 3A). Alternatively, transduced cells were cultured for 14 days inEpo/G-CSF/GM-CSF/SCF/IL-3/IL-6 to induce erythroid/myeloiddifferentiation (FIG. 3B). Cells were harvested and analyzed for GFPtransgene expression and cell-surface HLA-DR expression. FIG. 3demonstrates the strong promoter specificity of transgene expressionunder both differentiation conditions. While GFP expression is observedin roughly equal proportions in DR⁺ and DR⁻ progeny of CD34⁺ cellstransduced with the EF.GFP vector, GFP expression in the progeny ofCD34⁺ cells transduced with the DR promoter-driven lentiviral vector(DR.GFP) is limited to the DR⁺ cell subset (FIG. 3B). Both vectorsexpressed highly in DR⁺ cells after DC differentiation (FIG. 3A).

EXAMPLE 5 Transduced Human CD34⁺ Cells Can Engraft In Vivo and GenerateMultiple-Lineages of GFP+Cells Including DC That Are Functional inStimulating T Cell Proliferation

[0300] We next examined the DR-mediated transgene expression in humancells derived from transduced CD34⁺ cells post BMT in NOD/SCID mice. 10weeks post transplantation with transduced CD34⁺ cells, human progenyresiding in the BM and spleen of transplanted mice were analyzed for thepresence of human (CD45⁺) cells (FIG. 4A). Gated human cells werefurther analyzed for GFP, CD34 and MHC II expression (FIG. 4B). Weobserved that ˜30% of human cells in BM retained the CD34⁺ phenotype andthe majority of human cells displayed MHC II. In this experiment, ≧50%of the CD34⁺ or DR⁺ human cells expressed GFP in each engrafted mousewith DR.GFP-transduced cells (FIG. 4B). For EF.GFP-transduced cells, GFPexpression was observed in all the distinct human cell populationsincluding human CD34⁺, DR⁺, CD19⁺ (B lymphoid) and CD13⁺/CD33⁺ (myeloid)cells (data not shown). These results demonstrated that multipotent,engrafting HSC have been transduced. We further examined transgeneexpression in progeny derived from engrafted human cells that werecapable of differentiating into mature DCs (FIG. 4C). The DCs derivedfrom progenies of DR.GFP-transduced CD34⁺ cells indeed expressed highlevels of GFP, indicating their capacity for mediating gene expressionselectively in DCs. These differentiated DCs from engrafted humanprogenitors can potently stimulate the proliferation of allogeneic bloodlymphocytes.

Example 6 DR. GFP Vector Selectively Expresses a Transgene in MHC II⁺Mouse DC Differentiated from Lentiviral Transduced Mouse BM Cells

[0301] The human DR alpha promoter in the DR.GFP vector was also foundselectively-expressed in mouse MHC II⁺ cells (data not shown). Theconfirmed selective transgene expression of the DR.GFP vector allowed usto use the same vector for both human and mouse cells. We used these LVto transduce BM cells freshly isolated from normal BALB/c mice. After1-2 rounds of transduction, cells were cultured with GM-CSF to allow DCdifferentiation. At day 8 post DC differentiation, DC-enriched cellpopulations were analyzed by FACS for the expression of GFP transgeneand the I-E^(d) (MHC II) marker on the cell surface (FIG. 5). Over 17%of cells were transduced (GFP⁺) by either vector. In cells transduced byDR.GFP, GFP expression was selectively observed in MHC II⁺ cells. Thus,we confirmed the specificity of the DRalpha promoter-mediated transgeneexpression in MHC II⁺ cells.

EXAMPLE 7 Transduced Mouse BM HSC can Engraft Post BMT, and GenerateGFP⁺ DC

[0302] In order to set up a murine system to study the in vivoimmunologic effects of transduced HSC, we performed syngeneictransplantation in lethally irradiated BALB/c mice using DR.GFP- orEF.GFP-transduced mouse BM cells. At 10 weeks, BM and spleen cells wereharvested, analyzed and further cultured in GM-CSF to allow DCdifferentiation and maturation. Cells displayed a DC phenotype (FIG.6A), and were further analyzed for GFP expression vs the MHC II marker(FIGS. 6B and C). GFP expression by the DR.GFP vector was almostexclusively seen in the cells expressing the highest level of surfaceMHC II.

EXAMPLE 8 HA-Transduced HSC Post BMT Stimulate Massive Expansion ofHA-Specific CD4⁺ T Cells Following Adoptive Transfer into Recipient Mice

[0303] Having demonstrated the efficient transduction of both human andmurine HSC as well as significant expression of a marker transgene inthe HSC progeny subsequent to transplantation, we proceeded to evaluatethe capacity of this strategy to activate antigen-specific T cells. Forthese studies, we utilized HA as a model antigen because it is wellcharacterized and because we can follow HA-specific T cell responsesthrough the use of marked anti-HA TCR transgenic CD4 and CD8 cells (seeabove for more details). For these experiments, HA-expressing LV wereconstructed by replacing the GFP gene with the HA gene (DR.HA and EF.HA,FIG. 2). Prior to performing in vivo studies, we confirmed the abilityof these vectors to express functional HA in DCs cultured in vitro fromBM in GM-CSF cultures. FIG. 7 demonstrates that BM-derived DCstransduced with HA-expressing LV can greatly stimulate the proliferationof HA-specific T cells in vitro. Similar stimulation was obtained wheneither EF.HA or DR.HA were used (data not shown).

[0304] We then proceeded to test the capacity of DC derived fromtransplanted HA transduced HSC to stimulate HA specific T cells in vivo.BALB/c mouse BM cells were transduced with GFP- or HA-expressing LV.Three weeks after reconstituting irradiated BALB/c recipients with thetransduced HSC, animals were treated systemically with Flt3 ligand (FL)for 10 days in order to initiate in vivo DC expansion anddifferentiation. Eight days after initiation of in vivo FL treatment, 2million 6.5 T cells (anti-HA CD4 TCR-Tg line) were transferred iv.Activation of HA-specific T cells in vivo was monitored both physicallyand functionally. In a typical transfer experiment (no BMT or FLtreatment), 6.5 T cells represent a tiny proportion of the totalperipheral T cells (˜0.1%). Expansion of 6.5 T cells in vivo aftertransfer into mice that had received HA-LV transduced HSC was analyzedby double staining for CD4 and 6.5 clonotype at 5 and 8 days post T celltransfer (FIGS. 8A and 8B). At day 5 after transfer, a substantialexpansion of 6.5 cells was observed in animals transplanted with LV-HAtransduced HSC. 6.5 T cells expanded to 30% of total splenic CD4 cellsin animals transplanted with HSC transduced with EF.HA and DR.HArespectively. These numbers were significantly greater than in animalsreceiving control GFP-transduced HSC (4%). The expansion of 6.5 T cellsin animals receiving HA-transduced HSC was also significantly greaterthan animals vaccinated with recombinant vaccinia-HA (11%, data notshown), which in our hands is an extremely potent HA vaccine (Sotomayor,E. M. et al. (1999), PNAS 96:11476; and Sotomayor, E. M. et al. (1999),Nat. Med 5:780). The frequencies of HA-specific CD4⁺ T cells remainedhigh (7-10% vs 2% in the GFP control) when animals were examined 8 daysafter transfer (FIG. 8B). To determine whether this expansion in vivorepresented induction of effector function, we examined IFN-gammarelease by these T cells after in vitro culture with HA antigen. We andothers have previously shown that IFN-γ production correlates with Tcell activation to effector function and is significantly suppressedwhen antigen-specific tolerance is induced (Staveley-O'Carroll, K. etal. (1998), PNAS 95:1178; Sotomayor, E. M. et al. (1999), PNAS 96:11476;and Sotomayor, E. M. et al. (1999), Nat. Med 5:780). Indeed, we foundrobust IFN-γ production by 6.5 T cells from animals transplanted withHA-transduced HSC that was significantly higher than from the animalstransplanted with GFP-transduced control HSC (FIG. 8C). These resultsindicate that potent T cell activation is achieved with immunizationwith HA-transduced HSC engraftment and FL treatment.

[0305] As a parallel comparison group, BALB/c mice were immunized withex vivo differentiated (mature) DCs that were transduced withHA-expressing LV during ex vivo culture (the classic ex vivoDC-transduction/vaccination paradigm). Differentiated DCs weretransduced with EF.HA-expressing actively stimulated HA-specific T cellsin vitro (FIG. 7). However, multiple sc immunizations with these matureHA-transduced DCs induced a relatively modest expansion of HA-specific6.5 cells in vivo—far less than the level observed in animalstransplanted with HA-transduced HSC (FIG. 8D). These preliminary resultsindeed suggest that transduction of HSC followed by transplantation andin vivo DC differentiation may be much more efficient to activateantigen-specific T cells. Thus, even without in vivo DC activation byagents such as CD40, the strategy of transduction of HSC, followed bytransplantation and induction of in vivo DC differentiation, appears tobe an extremely promising approach to activate antigen-specific T cells.

[0306] Based on the data that DR promoter driven expression of HA maysomewhat enhance in vivo T cell expansion relative to a constitutivepromoter (EF1a), one could utilize other DC-specific promoters. Theseinclude the CIITA promoter 1, which was reported functional solely inDC; Mach, B. (1999), Science 285:1367; and/or the promoters of two novelDC specific genes (B7-DC and MINOR) that were recently discovered (seee.g. Tseng, S. Y. et al. (2001), J. Exp. Medicine, in press).

[0307] After HSC engraftment, patients may be treated systemically withvarious agents to promote DC differentiation in vivo. These includespecific cytokines, such as GM-CSF, FL, and/or an agonist anti-CD40antibody. Subsequent to these manipulations, mature T cells may betransferred into patients—the analogue of DLI that is now routinely usedin clinical BMT settings.

[0308] While systemic administrations of FL and CD40 agonists are potentin promoting DC expansion, differentiation and activation, they may alsobe potentially toxic to hosts. To overcome this problem and takeadvantage of the selective transgene expression in transduced cells byour novel LV system, bi-cistronic vectors in which a DC-specificpromoter expresses the tumor antigen and drug-inducible DC activatorgenes are created using standard methods of cloning known in the art(FIG. 2B) (see e.g. Molecular Cloning: A Laboratory Manual, secondedition (1989) by Sambrook, Fritsch and Maniatis; Cold Spring HarborLaboratory Press). The inducible DC activator is based on theintracellular (signal transducing) domain of CD40 linked to the FK506binding domain of FKBP12. Since 1993, several bivalent analogs of FK506such as FK1012 and AP1903 have been synthesized (see e.g. Spencer et al.(1993) Science 262: 1019; Amara et al. (1997) PNAS USA 94: 10618; andClackson et al. (1998) PNAS USA 95: 10437). These compounds are cellpermeable, immunologically inert and able to induce crosslinking andsignal transduction of chimeric molecules containing the FK506 bindingdomain and the signaling domain of various molecules includingc-kit/SCFR, Epo and Tpo. Accordingly, the invention may be adapted foruse with functional intracellular CD40-FKBP chimeric molecule, becausethis has already been validated with another member of the TNFR/CD40family, Fas. Amara, J. F. et al. (1997), PNAS 94:10618; Clackson, T. etal. (1998), PNAS 95:10437; and Thomis, D. C. et al. (2001), Blood97:1249. Specifically a chimeric gene is constructed with themembrane-anchoring domain of NGFR (p75) linked to tandem FKBP domainsfollowed by the cytoplasmic (signaling) domain of CD40. (see Clackson,T. et al. (1998), PNAS 95:10437; and Thomis, D. C. et al. (2001), Blood97:1249). The bi-cistronic vectors are then used to co-express theantigen gene and the NGFR-FKBPm2-iCD40 chimera gene controlled by aDC-specific promoter (FIG. 2B). The antigen and NGFR-FKBPm2-iCD40 areco-expressed specifically in differentiated DCs, and only thesetransduced donor DCs are activated intracellularly and quantitativelythrough the CD40 pathway after AP1903 administration at a given dose. Asecond functional intracellular-FKBP chimeric APC-stimulatory gene wouldbe the NGFR-FKBPm2-iFlt3 fusion protein which may obviate the need forsystemic FL administration for in vivo DC induction.

DETAILED DESCRIPTION OF THE FIGURES

[0309]FIG. 1 depicts lentiviral SIN vectors which allow high levels ofstable gene transfer and regulated transgene expression from a selectivepromoter in transduced cells. The U3 region in the prime LTR is replacedby a non-HIV promoter P1 to allow synthesis of viral genomic RNA inpackaging cells. The U3 region in the 3 prime LTR is largely deleted sothat it no longer functions as a promoter in taget cells postintegration. In transduced target cells, the transgene expressionmediated by these self-activating (SIN) vectors is solely controlled bythe internal promoter (P2) such as from the HLA-DRα (DR) gene. Thetiters of VSV-G pseudotyped viruses made by transfection in 293 T cellsare ˜1-6×10⁶ CFU/ml.”

[0310]FIG. 2 shows a number of lentiviral vectors expressing theselected antigen and the APC-stimulatory gene. FIG. 2 includes a list ofmono-cistronic lentiviral vectors we have made. The promoter from thehuman housekeeping gene, EF1a, is used as control. Lentiviral vectorsunder the control of a more DC-specific promoter (such as the CIITApromoter 1) are also included, (β), Bi-cistronic vectors are constructedunder the control of DR or a DC-specific promoter. DC activators areconstructed by fusing the intracellular signaling domain of CD40 or Flt3receptor with the FKBP domain. The engineered activator gene linked toe.g. the HA gene by an IRES are selectively expressed inside transducedDCs in a non-active form, which can be activated by a cell permeable,immunologically inert compound such as FK1012. Alternatively, constructvectors selectively expressing CD40 ligand or FLT3 ligand in transducedDC are constructed to generate an autocrine loop. A similar autocrine orparacrine loop can be achieved for the production of GM-CSF and IL-12 bytransduced DCs.

[0311]FIG. 3 shows that a GFP transgene driven by the DRα promoter isselectively expressed in human HLA-DR+cells differentiated fromCD34⁺cells transduced by the DR.GFPSIN lentiviral vector. HumanCD34⁺cells from G-CSF mobilized peripheral blood are cultured overnightand transduced once (as described in 3 a) with lentiviral vectors.Transgene expression is controlled by the promoter of either human DRαgene (DR.GFP vector) or a housekeeping gene such as EF1α promoter(EF.GFP vector). Transduced cells are cultured either in suspension withGM-CSF, IL-4 and TNFα to allow DC differentiation (A), or inmethylcellulose with Epo, G-CSF, GM-CSF SCF, IL-3, and IL-6 to allowerythroid and myelold colony formation (B). Cells differentiated undercondition A were harvested at day 7 post induction of differentiation.Cells differentiated under condition B were harvested after 14 days ofCFC assays. Cells were then analyzed by FACS for GFP and cell-surfaceHLA-DR expression. The HLA-Dr+ cells in (B) were likely to bemonocytes/macrophages derived from CFU-GM. In addition, we used bothlentiviral vectors to transduce non-dividing DC differentiated in vitrofrom human monocytes. We found that >20% of cells displayed DCphenotypes were readily transduced and conferred persistent GFPexpression at moderate levels.

[0312]FIG. 4 shows that transduced human CD34+ cells engraft in vivo andgenerate multiple lineages of GFP+hematopoietic cells including DCs thatcan stimulate allogeneic human T cell proliferation. Human cord bloodCD34+ were transduced either by DR.GFP as in FIG. 3. Transduced cellswere i.v. injected into conditioned NOD/SCID mice. Six to ten weeks postBMT, BM cells were were harvested from engrafted and the presence ofhuman (CD45+) donor cells (A). Human GFP+ cells that co-expressing CD34or HLA-DR (MHC II) markers were further analyzed (B). The DRα promoterdirected transgene expression exclusively in MHC II+ cells and in bothCD34+ and CD34+ cells. Similar results were obtained from human cellsengrafted in mouse spleen, except percentages of CD45+ and CD34+ werelower (data not shown). Engrafted human cells from mouse BM werepurified (by depleting mouse cells) and cultured subsequently under DCdifferentiation conditioned as in FIG. 3A. These differentiated DCsafter subsequent culture were analyzed by FACS(C and D). TheDR.GFP-transduced cells showed highly selective expression in MHCII^(high) cells after DC differentiation and maturation (C). Theirmorphology and phenotypes (D) and their functional activities instimulating allogenic human T cells (E) were also determined. Thepotency of these BM or splenic (SP) DCs post BMT following genetransduction (by either DR.GFP or EF.GFP LV) was high in this in introassay, and similar to those (CB CD34−DC) derived directly from CD 34+cells without BMT (E).

[0313]FIG. 5 shows that a GFP transgene driven by the DRα promoter isselectively expressed in mouse MHC II+ cells differentiated from bonemarrow cells transduced by the DR.GFP SIN lentiviral vector. After oneround of lentiviral transduction, mouse BM cells were cultured withGM-CSF and 10% FBS. Cells in suspension were discarded and adherentcells were fed at day 2 and 4. At day 6 adherent cells were transferredto new culture plates. Suspension cells were further selected at day 7and harvested at day 8 post differentiation. The DC-enriched cellpopulations were analyzed by FACS for GFP and cell-surface I-E (MHCII)expression. The sub-populations with the highest level of cell-surfaceI-E expression were known to be DC whereas the sub-populations withmedium to low levels of the cell-surface IE expression were immature DCand/or monocytes/macrophages. Intracellular MHC II molecules weredetected in the latter population. Note that the DR.GFP vector did notexpress in cells lacking cell-surface I-E (MHC II) expression.

[0314]FIG. 6 shows selective transgene expression mediated by the DRαpromoter in mouse donor-derived DCs post transduction and BMT. MouseHSCs harvested from BM of BALB/c mice (without 5-FU pretreatment) wereenriched by depleting mature cells expressing various hematopoieticlineage markers. After overnight culture, stimulated cells weretransduced by VSV-G pseudotyped DR.GFP LV. After 1-2 rounds oftransduction, cells were either infected into lethally irradiated BALB/cmice or culture in vitro (with GM-CSF) for DC differentiation (as shownin FIGS. 5 and 7). Six to ten weeks after transplant, BM or spleniccells harvested from engrafted mice were further analyzed for their DCcompartment. Splenic DCs were enriched and activated by overnightculture. The phenotypes and transgene expression are shown in (A) and(B), respectively. (C): BM cells from engrafted BALF/c mice were furtherdifferentiated into DCs as in FIG. 5. Note that either donor engraftmentor gene transduction rate, judged by percentages of GFP+ cells, isunderestimated, since it is unlikely that we transduced all the donorcells.

[0315]FIG. 7 shows mouse DCs differentiated from transduced BM cells bythe DR.HA lentiviral vector can specifically and potently stimulate theproliferation of HA-specific T cells (from the 6.5 HA-TCR Tg mice).

[0316]FIG. 8 HA shows transduced HSCs post BMT greatly stimulate theproliferation of HA-specific CD4+ T cells and IFN-y production followingadoptive T cell transfer. This illustrates the ability ofDR.HA-transduced HSCs post BMT to initiate HA-specific immunue responsesafter T cell transfer from the 6.5 HA-TCR Tg mice, 3 weeks post BMT withGFP- or HA-transduced HSC. Receipient mice were treated with FL dailyfor 10 days to expand and stimulate DC. At day 8, 6.5 T cells (2.5 g⁸)were iv transferred. Frequencies of the 6.5 clongenic CD+ T cells inspleen were measured by FACS with anti-6.5 TCR ad anti-CD4 antibodies atday 5(A) or (B) after T cell transfer. CD4− cells are shown andpercentage of 6.5 clonotypic cells among total CD4+ T cells areindicated (A and B). IFN-γ production of harvested T cells at day 8 werealso measured (C). Harvested splenocytes from animals engrafted withGFP-transduced or HA-transduced HSC were incubated with naive BM-derivedDC (as APCs) pulsed with the HA (Class II-restricted) peptide. Thereleased IFN-γ in the first 24 hours measured by ELISA. Average ofduplicated samples were plotted.

[0317] Equivalents

[0318] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims.

1. A method of modulating a T cell-dependent immune response to anantigen in a mammalian host comprising: providing a population ofhematopoietic stem cells; introducing into said population ofhematopoietic stems cells a first gene, comprising the antigen, and asecond gene, comprising a factor that regulates antigen presenting celldifferentiation, maturation, expansion or activation to create apopulation of transgenic cells; transplanting said transgenic cells intothe mammalian host; and allowing said transgenic cells to develop invivo into antigen presenting cells that express said antigen, therebymodulating a T cell-dependent immune response to the antigen in themammalian host.
 2. The method of claim 1, wherein the population ofhematopoietic stem cells are provided from an autologous bone marrowgraft.
 3. The method of claim 1, wherein the population of hematopoieticstem cells are provided from an allogeneic bone marrow graft.
 4. Themethod of claim 1, wherein the population of hematopoietic stem cellsare provided from an xenogeneic bone marrow graft.
 5. The method ofclaim 1, wherein the antigen is a tumor antigen.
 6. The method of claim5, wherein the tumor antigen is selected from the group consisting of: aprostate-specific membrane antigen (PSMA), a HER2/neu gene antigen, anidiotypic immunoglobulin antigen, an idiotypic T cell receptor antigen,an SV40 antigen, and a carcinoembryonic antigen (CEA).
 7. The method ofclaim 1, wherein the antigen is a immune tolerance-inducing antigen. 8.The method of claim 7, wherein the immune tolerance-inducing antigen isselected from the group consisting of: pancreatic beta-cell antigens,insulin, GAD, collagen type 11, human cartilage gp 39 (HCgp39),gp130-RAPS, myelin basic protein (MBP), proteolipid protein (PLP),myelin oligodendrocyte glycoprotein, fibrillarin, small nucleolarprotein (snoRNP), thyroid stimulating factor receptor (TSH-R), histones,glycoprotein gp70, ribosomal protein, pyruvate dehydrogenasedehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigens andhuman tropomyosin isoform 5 (hTM5).
 9. The method of claim 1, whereinthe antigen is a pathogen antigen.
 10. The method of claim 9, whereinthe pathogen antigen is selected from the group consisting of: ahepatitis B antigen, a tuberculosis antigen, an HIV antigen, and aBorrelia burgdorferi sensu lato antigen.
 11. The method of claim 1,wherein the factor that regulates antigen presenting celldifferentiation, maturation, expansion or activation is selected fromthe group consisting of: iCD40-FKBP; iFlt3-FKBP; CD40 ligand; Flt3ligand; GM-CSF; and IL-12.
 12. The method of claim 1, wherein said firstgene comprising the antigen further comprises a dendritic cell-specificpromoter.
 13. The method of claim 12, wherein the dendriticcell-specific promoter is selected from the group consisting of: an EF1apromoter; an HLA-DR promoter; a CIITA P1 promoter; a B7-DC promoter; anda minor gene promoter.
 14. The method of claim 1, wherein the transgeniccells are transplanted into the mammalian host before allowing thetransgenic cells to develop into antigen presenting cells that expressthe antigen.
 15. The method of claim 1, wherein the transgenic cells aretransplanted into the mammalian host after allowing the transgenic cellsto develop into antigen presenting cells that express the antigen. 16.The method of claim 1, wherein modulation of the T cell-dependent immuneresponse effects an immunization against a viral or microbial pathogen.17. The method of claim 1, wherein modulation of the T cell-dependentimmune response effects an immune response against a tumor antigen. 18.The method of claim 1, wherein modulation of the T cell-dependent immuneresponse effects immune tolerance for an autoantigen.
 19. A method ofmodulating a T cell-dependent immune response to an antigen in amammalian host comprising: providing a population of hematopoietic stemcells; introducing into said population of hematopoietic stems cells afirst gene, comprising the antigen to create a population of transgeniccells; contacting said transgenic cells with a factor that regulatesantigen presenting cell differentiation, maturation, expansion oractivation; transplanting said transgenic cells into the mammalian host;and allowing said transgenic cells to develop in vivo into antigenpresenting cells that express said antigen, thereby modulating a Tcell-dependent immune response to the antigen in the mammalian host. 20.The method of claim 19, wherein the population of hematopoietic stemcells are provided from an autologous bone marrow graft.
 21. The methodof claim 19, wherein the population of hematopoietic stem cells areprovided from an allogeneic bone marrow graft.
 22. The method of claim19, wherein the population of hematopoietic stem cells are provided froman xenogeneic bone marrow graft.
 23. The method of claim 19, wherein theantigen is a tumor antigen.
 24. The method of claim 23, wherein thetumor antigen is selected from the group consisting of: aprostate-specific membrane antigen (PSMA), a HER2/neu gene antigen, anidiotypic immunoglobulin antigen, an idiotypic T cell receptor antigen,an SV40 antigen, and a carcinoembryonic antigen (CEA).
 25. The method ofclaim 19, wherein the antigen is an immune tolerance-inducing antigen.26. The method of claim 25, wherein the immune tolerance-inducingantigen is selected from the group consisting of: pancreatic beta-cellantigens, insulin, GAD, collagen type 11, human cartilage gp 39(HCgp39), gp130-RAPS, myelin basic protein (MBP), proteolipid protein(PLP), myelin oligodendrocyte glycoprotein, fibrillarin, small nucleolarprotein (snoRNP), thyroid stimulating factor receptor (TSH-R), histones,glycoprotein gp70, ribosomal protein, pyruvate dehydrogenasedehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigens andhuman tropomyosin isoform 5 (hTM5).
 27. The method of claim 19, whereinthe antigen is a pathogen antigen.
 28. The method of claim 27, whereinthe antigen is a pathogen antigen selected from the group consisting of:29. The method of claim 19, wherein the factor that regulates antigenpresenting cell differentiation, maturation, expansion or activation isselected from the group consisting of: iCD40-FKBP; iFlt3-FKBP; CD40ligand; Flt3 ligand; GM-CSF; and IL-12.
 30. The method of claim 19,wherein said first gene comprising the antigen further comprises adendritic cell-specific promoter.
 31. The method of claim 30, whereinthe dendritic cell-specific promoter is selected from the groupconsisting of: an EF1a promoter; an HLA-DR promoter; a CIITA P1promoter; a B7-DC promoter; and a Minor gene promoter.
 32. The method ofclaim 19, wherein the transgenic cells are transplanted into themammalian host before allowing the transgenic cells to develop intoantigen presenting cells that express the antigen.
 33. The method ofclaim 19, wherein the transgenic cells are transplanted into themammalian host after allowing the transgenic cells to develop intoantigen presenting cells that express the antigen.
 34. The method ofclaim 19, wherein modulation of the T cell-dependent immune responseeffects an immunization against a viral or microbial pathogen.
 35. Themethod of claim 19, wherein modulation of the T cell-dependent immuneresponse effects an immune response against a tumor antigen.
 36. Themethod of claim 19, wherein modulation of the T cell-dependent immuneresponse effects immune tolerance for an autoantigen.
 37. A vectorexpression system comprising: a first gene expression cassettecomprising an antigen gene under control of an antigen presentingcell-specific promoter; and a second gene expression cassette comprisinga factor gene that stimulates antigen presenting cell differentiation,maturation, expansion or activation.
 38. The vector expression system ofclaim 37, wherein the vector expression system is a lentiviral vector.39. The vector expression system of claim 37, wherein the antigenpresenting cell-specific promoter is selected from the group consistingof: an HLA-DR promoter; a CIITA P1 promoter; a B7-DC promoter; and aMinor gene promoter.
 40. The vector expression system of claim 37,wherein the first gene expression cassette comprises a pathogen antigengene selected from the group consisting of: a hepatitis B antigen, atuberculosis antigen, an HIV antigen, and a Borrelia burgdorferi sensulato antigen.
 41. The vector expression system of claim 37, wherein thefirst gene expression cassette comprises a tumor antigen selected fromthe group consisting of: a prostate-specific membrane antigen (PSMA), aHER2/neu gene antigen, an idiotypic immunoglobulin antigen, an idiotypicT cell receptor antigen, an SV40 antigen, and a carcinoembryonic antigen(CEA).
 42. The vector expression system of claim 37, wherein the immunetolerance-inducing antigen is selected from the group consisting of:pancreatic beta-cell antigens, insulin, GAD, collagen type 11, humancartilage gp 39 (HCgp39), gp130-RAPS, myelin basic protein (MBP),proteolipid protein (PLP), myelin oligodendrocyte glycoprotein,fibrillarin, small nucleolar protein (snoRNP), thyroid stimulatingfactor receptor (TSH-R), histones, glycoprotein gp70, ribosomal protein,pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2), hairfollicle antigens, and human tropomyosin isoform 5 (hTM5).
 43. Thevector expression system of claim 37, wherein the factor gene thatstimulates antigen presenting cell differentiation, maturation,expansion or activation is selected from the group consisting of:iCD40-PKBP; iFlt3-FKBP; CD40 ligand; Flt3 ligand; GM-CSF; and IL-12.